**3.4 Nonlinear chromatography for determination of kinetics parameters**

approach can be used to calculate the association rate constant (*kon*), dissociation rate constant (*koff*) for the ligand–receptor complex and the equilibrium constant for complex formation (*K*). One approach to the analysis of NLC data is the Impulse Input Solution for the mass balance equation developed in 1987 by Wade and collaborators (Wade et al., 1987, as cited in Moaddel & Wainer, 2006). This approach is based upon the observation that when adsorption/desorption rates are slow, band broadening is insensitive to a moderate degree of column overload. In contrast to numerical integration methods this approach uses the analytical solution, which can be applied directly to t experimental peak proles. The Impulse Input Equation has been included in commercially available deconvolution software, and can be easily applied to NLC studies. The peak area parameter (*a0*), peak center parameter (*a1*), its width (*a2*) and distortion (*a3*) are the parameters used to describe the chromatographic traces. Thermodynamic and kinetics parameters of a drug-receptor complex formation are then calculated on the basis of the relationship:

*k'* = *a1* the real thermodynamic capacity factor

*kd = 1/a2t0* desorption reaction rate constant

*Ka = a3/C0* adsorption equilibrium constants, where *C0* is the concentration of solute injected multiplied by the width of the injection pulse (as a fraction of column dead volume)

*ka* = *kd Ka* reaction rate constant of adsorption

### **4. Examples of application**

QAC was successfully used to describe drug interactions with multiple different system proteins. In case of soluble proteins this approach was applied to determine binding of different active substances (drugs, hormones) with e.g. serum albumins (HSA), α1-acid glycoprotein (AGP) or nucleic estrogen receptors (hER).

Xuan & Hage (2005) conducted research on immobilized α1-acid glycoprotein (AGP) demonstrating good correlation (0.954) of observed retention factors of several compounds with their equilibrium binding constants to AGP designated by other methods. In the same work authors, using the self-competition technique, confirmed the literature data on existence of one binding site for propranolol enantiomers on this protein. Determined values of *Ka* were also included in the range of values known from other experiments. Performing the analysis at different temperatures, changes in enthalpy and entropy of propranolol enantiomers binding to α1-acid glycoprotein, and their contribution in the total energy change in the process of binding were determined. It was found, that this reaction depends mainly on the enthalpy, but entropy change also significantly affects the binding of propranolol. Linearity of the plot confirmed the single-side character of binding of this compound by the AGP.

Combination of zonal QAC with competition-displacement studies confirmed the negative allosteric nature of the interaction between verapamil and tamoxifen. It was determined that verapamil causes 41% decrease in binding constant of tamoxifen by immobilized HSA (Malik et al., 2008). As mentioned earlier, using compound of interest as a competitive addition to the mobile phase it is possible to define its interactions with target protein on a single binding site even though the drug is bound to several different sites on the protein.

Affinity Chromatography as a Tool for Quantification of

and the binding site on the nAChR.

Interactions Between Drug Molecules and Their Protein Targets 293

QAC was also used in determination of binding site structure of the α3β4 nAChR. Comparing the retention time of tritium labelled epibatidine ([3H] EB) on four different columns: with immobilized either receptor subunit α3 or β4 subunit, both subunits immobilized on the same column and complete pentameric α3β4 receptor, it was found that the binding site for this compound is formed by several subunits of the complete receptor, and not on the individual subunits. This was confirmed by assays with nicotine as a competitive additive to the mobile phase. No impact of nicotine on the retention of [3H] EB on columns with immobilized subunits was observed, while the epibatidine retention on the column with complete receptor decreased from k = 8.4 to k = 1.5, which indicates the interaction of nicotine on [3H] EB binding site (Wainer et al., 1999). In the same work the effect of pH and ionic strength of mobile phase on the retention of [3H] epibatidine in the α3β4 nAChR column was also examined using zonal elution technique. Analyses showed an increase in epibatidine retention with increasing mobile phase pH from pH 4 to pH 7 and a significant decrease in the retention of this compound with increasing ionic strength of mobile phase (5-200 mM ammonium acetate concentration). These results showed that the binding of competitive agonists is mainly electrostatic interaction between drug molecule

QAC in combination with displacement studies were used in case of α3β4 nAChR to confirm the location of binding sites of allosteric inhibitors of this receptor. Elution of bupropion, ketamine and dextromethorphan was conducted in the presence of mecamylamine (nAChR blocker) as an addition to the mobile phase. The linear dependence of eluted compounds 1/*k'* on the displacer concentration indicates that all tested inhibitors compete for the same binding site on the receptor molecule. Additional studies also showed no effect of mecamylamine in the mobile phase on epibatidine retention and no effect of nicotine in the mobile phase on the retention of ketamine and bupropion. It indicates that ketamine, bupropion, mecamylamine bind to other sites on the receptor surface than nicotine and epibatidine and do not compete for the binding site (Jozwiak et al., 2002). In the same work nonlinear chromatography (NLC) model was used to analyse interactions between the immobilized α3β4 nAChR, and its allosteric inhibitors. Peak profiles from zonal elution of allosteric inhibitors (ketamine, bupropion, mecamylamine, and dextrometorfan), recorded by the mass spectrometry were numerically fitted to Impulse Input Solution model using PeakFit v4.11 software (SPSS Inc., Chicago, IL). The results (*kon*, *koff*, *K*) were consistent with available literature data, thus confirming the effectiveness of the NLC in receptor drug binding kinetics study, in this case allosteric nAChR inhibitors. In further work enantioselectivity of the interaction of dextromethorphan (DM) and levomethorphan (LM) with an immobilized α3β4 nAChR subtype liquid chromatographic stationary phase has been compared to DM- and LM-induced non-competitive blockade of nicotine-stimulated 86Rb+ efflux from cells expressing the α3β4 nAChR. Since DM and LM are enantiomers and have the same physicochemical properties, any chromatographic and pharmacological differences must be due to specific interactions with nAChR. Asymmetrical peaks were observed for both compounds and DM had signicantly longer retention time than LM (Figure 8). Determined by NLC *kon* values of both compounds did not significantly differ while *koff* of DM was significantly lower than *koff* value of LM. That was in good agreement with results of the functional inhibition studies which showed that DM and LM had equivalent potencies, i.e. the same IC50 values, but that DM inhibition lasted longer than the effect produced by LM. The effect of temperature on the chromatographic retention of DM

Such solution has been used in the study of binding of both; hormones (Loun & Hage, 1995) and drugs (Chen et. al., 2004; Yoo et al., 2009; Mallik et al., 2008) to different places in the HSA molecule. Simultaneous injection of imipramine with L-tryptophan on the column with immobilized HSA, showed a competitive interaction between those two drugs, confirming that imipramine specifically connects to the indole-benzodiazepine binding site on the molecule of albumin (Sudlow site II). This analysis also allowed to determine the equilibrium constant of this interaction (Yoo et al., 2009). Analogously was defined binding site for the verapamil on this protein, demonstrating in this case, competitive interaction with warfarin, specifically connecting to Sudlow site I. This was confirmed by elution in the presence of tamoxifen. Nonlinear dependence of retention of this compound on verapamil concentration showed a high correlation to the equation describing the allosteric interaction, characteristic for interaction that is observed between the tamoxifen binding site and the warfarin binding site (Sudlow I) (Mallik et al., 2008). Team of Chen and colleagues (2004) determined the nature of the interaction of phenytoin by drawing a map of interactions of the drug with the major binding sites on the surface of HSA on the basis of the relationship between 1/*k* of the compounds with confirmed specific binding site on albumin molecule, and various concentrations of phenytoin as a mobile phase additive during elution. Thus suggested the potential impact of other concomitant medications on the efficacy of therapy with phenytoin.

The effect of different assay conditions on drug-protein interaction was used, for example to define the influence of long-chain fatty acids concentration in the plasma and glycation of plasma transport proteins on the binding of sulfonylurea drugs in diabetes. Experiments were carried out using columns with immobilized HSA (Basiaga & Hage, 2010).

Recently Sanghvi and co-workers (2010) successfully immobilized the ligand binding domains of the estrogen related receptors ERRα and ERRγ onto the aminopropyl silica liquid chromatography stationary phase, as well as the surface of the open tubular capillaries, creating the ERR-silica and ERR-OT columns. Both types were characterized using frontal chromatographic techniques with diethylstibesterol and the binding affinities, expressed as *Kd* values, to the immobilized receptors were consistent with the literature data. Biochanin A, the ERRα agonist, was also used to further characterization of properties of the ERRα-silica column, and obtained *Kd* value was consistent with the previously reported data. The ERRγ-silica column was characterized using nonlinear chromatographic techniques using a series of tamoxifen derivatives (tamoxifen, 4-hydroxy tamoxifen and Ndesmethyl 4-hydroxytamoxifen, Endoxifen). The relative *Kd* values obtained for the derivatives were consistent with relative ability of the compounds to inhibit the cellular proliferation of the human-derived T98G glioma cell line, expressed as IC50 values. The results indicate that the relative retention of compounds on these columns reflects the magnitude of their inhibitory activity. Therefore columns containing immobilized ERRs can be used for a preliminary screen for anti-glioma agents, such as tamoxifen, which work as selective estrogen receptor modulator and that this method may replace current laborious and time consuming cellular uptake studies.

QAC has been applied to study drug-receptor interactions in the α1 adrenergic receptor system. The results showed a positive correlation of chromatographic data (*k'*) with literature data defining the affinity of ligands on the basis of radio ligand studies with cell membrane homogenates containing α1 adrenergic receptor (Yu et al., 2005).

Such solution has been used in the study of binding of both; hormones (Loun & Hage, 1995) and drugs (Chen et. al., 2004; Yoo et al., 2009; Mallik et al., 2008) to different places in the HSA molecule. Simultaneous injection of imipramine with L-tryptophan on the column with immobilized HSA, showed a competitive interaction between those two drugs, confirming that imipramine specifically connects to the indole-benzodiazepine binding site on the molecule of albumin (Sudlow site II). This analysis also allowed to determine the equilibrium constant of this interaction (Yoo et al., 2009). Analogously was defined binding site for the verapamil on this protein, demonstrating in this case, competitive interaction with warfarin, specifically connecting to Sudlow site I. This was confirmed by elution in the presence of tamoxifen. Nonlinear dependence of retention of this compound on verapamil concentration showed a high correlation to the equation describing the allosteric interaction, characteristic for interaction that is observed between the tamoxifen binding site and the warfarin binding site (Sudlow I) (Mallik et al., 2008). Team of Chen and colleagues (2004) determined the nature of the interaction of phenytoin by drawing a map of interactions of the drug with the major binding sites on the surface of HSA on the basis of the relationship between 1/*k* of the compounds with confirmed specific binding site on albumin molecule, and various concentrations of phenytoin as a mobile phase additive during elution. Thus suggested the potential impact of other concomitant medications on the efficacy of therapy

The effect of different assay conditions on drug-protein interaction was used, for example to define the influence of long-chain fatty acids concentration in the plasma and glycation of plasma transport proteins on the binding of sulfonylurea drugs in diabetes. Experiments

Recently Sanghvi and co-workers (2010) successfully immobilized the ligand binding domains of the estrogen related receptors ERRα and ERRγ onto the aminopropyl silica liquid chromatography stationary phase, as well as the surface of the open tubular capillaries, creating the ERR-silica and ERR-OT columns. Both types were characterized using frontal chromatographic techniques with diethylstibesterol and the binding affinities, expressed as *Kd* values, to the immobilized receptors were consistent with the literature data. Biochanin A, the ERRα agonist, was also used to further characterization of properties of the ERRα-silica column, and obtained *Kd* value was consistent with the previously reported data. The ERRγ-silica column was characterized using nonlinear chromatographic techniques using a series of tamoxifen derivatives (tamoxifen, 4-hydroxy tamoxifen and Ndesmethyl 4-hydroxytamoxifen, Endoxifen). The relative *Kd* values obtained for the derivatives were consistent with relative ability of the compounds to inhibit the cellular proliferation of the human-derived T98G glioma cell line, expressed as IC50 values. The results indicate that the relative retention of compounds on these columns reflects the magnitude of their inhibitory activity. Therefore columns containing immobilized ERRs can be used for a preliminary screen for anti-glioma agents, such as tamoxifen, which work as selective estrogen receptor modulator and that this method may replace current laborious

QAC has been applied to study drug-receptor interactions in the α1 adrenergic receptor system. The results showed a positive correlation of chromatographic data (*k'*) with literature data defining the affinity of ligands on the basis of radio ligand studies with cell

membrane homogenates containing α1 adrenergic receptor (Yu et al., 2005).

were carried out using columns with immobilized HSA (Basiaga & Hage, 2010).

with phenytoin.

and time consuming cellular uptake studies.

QAC was also used in determination of binding site structure of the α3β4 nAChR. Comparing the retention time of tritium labelled epibatidine ([3H] EB) on four different columns: with immobilized either receptor subunit α3 or β4 subunit, both subunits immobilized on the same column and complete pentameric α3β4 receptor, it was found that the binding site for this compound is formed by several subunits of the complete receptor, and not on the individual subunits. This was confirmed by assays with nicotine as a competitive additive to the mobile phase. No impact of nicotine on the retention of [3H] EB on columns with immobilized subunits was observed, while the epibatidine retention on the column with complete receptor decreased from k = 8.4 to k = 1.5, which indicates the interaction of nicotine on [3H] EB binding site (Wainer et al., 1999). In the same work the effect of pH and ionic strength of mobile phase on the retention of [3H] epibatidine in the α3β4 nAChR column was also examined using zonal elution technique. Analyses showed an increase in epibatidine retention with increasing mobile phase pH from pH 4 to pH 7 and a significant decrease in the retention of this compound with increasing ionic strength of mobile phase (5-200 mM ammonium acetate concentration). These results showed that the binding of competitive agonists is mainly electrostatic interaction between drug molecule and the binding site on the nAChR.

QAC in combination with displacement studies were used in case of α3β4 nAChR to confirm the location of binding sites of allosteric inhibitors of this receptor. Elution of bupropion, ketamine and dextromethorphan was conducted in the presence of mecamylamine (nAChR blocker) as an addition to the mobile phase. The linear dependence of eluted compounds 1/*k'* on the displacer concentration indicates that all tested inhibitors compete for the same binding site on the receptor molecule. Additional studies also showed no effect of mecamylamine in the mobile phase on epibatidine retention and no effect of nicotine in the mobile phase on the retention of ketamine and bupropion. It indicates that ketamine, bupropion, mecamylamine bind to other sites on the receptor surface than nicotine and epibatidine and do not compete for the binding site (Jozwiak et al., 2002). In the same work nonlinear chromatography (NLC) model was used to analyse interactions between the immobilized α3β4 nAChR, and its allosteric inhibitors. Peak profiles from zonal elution of allosteric inhibitors (ketamine, bupropion, mecamylamine, and dextrometorfan), recorded by the mass spectrometry were numerically fitted to Impulse Input Solution model using PeakFit v4.11 software (SPSS Inc., Chicago, IL). The results (*kon*, *koff*, *K*) were consistent with available literature data, thus confirming the effectiveness of the NLC in receptor drug binding kinetics study, in this case allosteric nAChR inhibitors. In further work enantioselectivity of the interaction of dextromethorphan (DM) and levomethorphan (LM) with an immobilized α3β4 nAChR subtype liquid chromatographic stationary phase has been compared to DM- and LM-induced non-competitive blockade of nicotine-stimulated 86Rb+ efflux from cells expressing the α3β4 nAChR. Since DM and LM are enantiomers and have the same physicochemical properties, any chromatographic and pharmacological differences must be due to specific interactions with nAChR. Asymmetrical peaks were observed for both compounds and DM had signicantly longer retention time than LM (Figure 8). Determined by NLC *kon* values of both compounds did not significantly differ while *koff* of DM was significantly lower than *koff* value of LM. That was in good agreement with results of the functional inhibition studies which showed that DM and LM had equivalent potencies, i.e. the same IC50 values, but that DM inhibition lasted longer than the effect produced by LM. The effect of temperature on the chromatographic retention of DM

Affinity Chromatography as a Tool for Quantification of

substrate should be normalized according to the formula 11.

with covalently bounded arginase (André et al., 2011).

bed volume of the IMER.

**5. Conclusions**

Interactions Between Drug Molecules and Their Protein Targets 295

Attractive features of immobilized enzyme reactors are the increased enzyme stability and the reusability coupled to accuracy, automation and potential high throughput when they are inserted in a HPLC system (Bartolini et al., 2005). The approach requires neither highly purified enzyme nor use of labelled substrates (radio- or color labelled). In immobilized enzymes, inhibitors efficacies can be measured either as IC50 values or *Ki* values using numerical transformation. Enzyme kinetic parameters are determined by successive injection of a substrate at increasing concentrations and measuring the rate of enzymatic reaction (*V*) expressed as peak area of product formed after each injection. Fitting the data to Lineweaver–Burk double-reciprocal plot of 1/(*V*) against the substrate concentration *[S]* (what is a linear transformation of the Michaelis–Menten plot), *Km* and *Vmax* values can be obtained. The y-intercept of such graph is equivalent to the inverse of 1/Vmax, the x-intercept of the graph represents -1/Km. In order to obtain correct results the concentration of

> *C V inj inj S = BV*

where *Cinj* is the injected substrate concentration, *Vinj* is the injected volume and *BV* is the

To determine the inhibition constant (*Ki*) for a test drug a set of inhibitor injections in several different concentrations (*[I]*) at two or more concentration of a substrate should be performed. As noted by Dixon in 1953, if 1/*V* is plotted against inhibitor concentration *[I]*, a straight line plot is obtained for each substrate concentration *[S]*. The *[I]* value of a intersect of those lines is equal to -*Ki*. If curves obtained for several different *[S]* converge in the left upper quadrant of a chart, the inhibitor is competitive. If curves converge on the *[I]* axis, the

Simultaneous injections of both a substrate at a xed saturating concentration and increasing concentration of an inhibitor, result in increasing reduction of the peak area (*Ai*) in comparison to area obtained for a substrate alone (*A0*). The percent inhibition (100 - (*Ai*/*A0* x 100)) is then plotted against the inhibitor concentration to obtain the inhibition curves (Girelli & Mattei, 2005; Nie & Wang, 2009). Recently this technique has been used for the kinetic characterization of inhibitors specific to brain-targeted butyrylcholinesterase (BuChE) (Bartolini et al., 2009), acetylocholinesterase (AchE) (Bartolini et al., 2004; Bartolini et al., 2005; He et al., 2010) and βsecretase (human recombinant β-amyloid precursor protein cleaving enzyme, hrBACE1)(Mancini & Andrisano, 2010) as potential therapeutics for Alzheimer's disease. IMER was also used for rapid and cost-effective on-line chromatographic screening of matrix metalloproteinases (MMP-9 (Ma & Chun Yong Chan, 2010) and MMP-8 (Mazzini et al., 2011)) inhibitors that may be useful in cancer therapy and for determining the role of some derived plant products for treating NO-dependent smooth disorders using monolithic micro-IMER

The drive to bring innovative drugs to market faster, without compromising quality and safety, induced need for new experimental techniques and methodologies. It is crucial to

inhibitor is non-competitive. For not competitive inhibition, the lines are parallel.

(11)

and LM on the nAChR column was also determined using a sequence of temperature experiments ranging from 5 to 30°C. Respective *k*'s values of independently injected compound were determined and van't Hoff plots were constructed by plotting ln *k* versus 1/*T*. Results showed that the binding reaction is enthalpy driven. In addition, for DM and LM there was no significant difference in the *ΔS*° values, while the *ΔH*° value for the DMnAChR interaction was significantly lower than respective value for LM-nAChR interaction showing that the enantioselectivity of α3β4 nAChR is mostly enthalpy-based (Jozwiak et al., 2003).The results of those studies demonstrated that non-linear chromatography approach of investigations of immobilized nAChRs can be useful in characterizing ligand–receptor binding interactions and predicting of properties of drugs and drug candidates. Additional NLC studies with this system were used to construct QSAR models of non-competitive inhibitors of the nAChR (Jozwiak et al., 2004), to develop a molecular model of these interactions (Jozwiak et al., 2004) and to predict IC50 values (Jozwiak et al., 2005)

Fig. 8. The comparison of peak profiles of DM and LM obtained in independent experiments of consequent injections. Reprinted from *Journal of Chromatography B*, Vol. 797, No. 1-2, Jozwiak, K., Hernandez, S. C., Kellar, K. J., & Wainer, I. W. (2003). Enantioselective interactions of dextromethorphan and levomethorphan with the α3β4-nicotinic acetylcholine receptor: comparison of chromatographic and functional data, pp. 373-379, Copyright 2011, with permission from Elsevier

Competition displacement studies with [3H]epibatidine and [125I]α-bungarotoxin as marker radioligands specific for α3β4 and α7 nAChRs subtypes respectively were applied to determine *Ki* values and check subtype selectivity of newly synthesized derivatives of epiboxidine, synthetic epibatidine-related compounds (Rizzi et al., 2008).

Another interesting application of QAC is use of immobilized-enzyme reactors (IMERs). In medicinal chemistry research IMERs are applied to drug metabolism studies, enantioselective analyses and for the identification of substrates and inhibitors as potential drugs. Interestingly, the investigated enzymatic reaction took place directly on the column.

and LM on the nAChR column was also determined using a sequence of temperature experiments ranging from 5 to 30°C. Respective *k*'s values of independently injected compound were determined and van't Hoff plots were constructed by plotting ln *k* versus 1/*T*. Results showed that the binding reaction is enthalpy driven. In addition, for DM and LM there was no significant difference in the *ΔS*° values, while the *ΔH*° value for the DMnAChR interaction was significantly lower than respective value for LM-nAChR interaction showing that the enantioselectivity of α3β4 nAChR is mostly enthalpy-based (Jozwiak et al., 2003).The results of those studies demonstrated that non-linear chromatography approach of investigations of immobilized nAChRs can be useful in characterizing ligand–receptor binding interactions and predicting of properties of drugs and drug candidates. Additional NLC studies with this system were used to construct QSAR models of non-competitive inhibitors of the nAChR (Jozwiak et al., 2004), to develop a molecular model of these

interactions (Jozwiak et al., 2004) and to predict IC50 values (Jozwiak et al., 2005)

Fig. 8. The comparison of peak profiles of DM and LM obtained in independent experiments of consequent injections. Reprinted from *Journal of Chromatography B*, Vol. 797, No. 1-2, Jozwiak, K., Hernandez, S. C., Kellar, K. J., & Wainer, I. W. (2003). Enantioselective interactions of dextromethorphan and levomethorphan with the α3β4-nicotinic

acetylcholine receptor: comparison of chromatographic and functional data, pp. 373-379,

epiboxidine, synthetic epibatidine-related compounds (Rizzi et al., 2008).

Competition displacement studies with [3H]epibatidine and [125I]α-bungarotoxin as marker radioligands specific for α3β4 and α7 nAChRs subtypes respectively were applied to determine *Ki* values and check subtype selectivity of newly synthesized derivatives of

Another interesting application of QAC is use of immobilized-enzyme reactors (IMERs). In medicinal chemistry research IMERs are applied to drug metabolism studies, enantioselective analyses and for the identification of substrates and inhibitors as potential drugs. Interestingly, the investigated enzymatic reaction took place directly on the column.

Copyright 2011, with permission from Elsevier

Attractive features of immobilized enzyme reactors are the increased enzyme stability and the reusability coupled to accuracy, automation and potential high throughput when they are inserted in a HPLC system (Bartolini et al., 2005). The approach requires neither highly purified enzyme nor use of labelled substrates (radio- or color labelled). In immobilized enzymes, inhibitors efficacies can be measured either as IC50 values or *Ki* values using numerical transformation. Enzyme kinetic parameters are determined by successive injection of a substrate at increasing concentrations and measuring the rate of enzymatic reaction (*V*) expressed as peak area of product formed after each injection. Fitting the data to Lineweaver–Burk double-reciprocal plot of 1/(*V*) against the substrate concentration *[S]* (what is a linear transformation of the Michaelis–Menten plot), *Km* and *Vmax* values can be obtained. The y-intercept of such graph is equivalent to the inverse of 1/Vmax, the x-intercept of the graph represents -1/Km. In order to obtain correct results the concentration of substrate should be normalized according to the formula 11.

$$\begin{bmatrix} S \end{bmatrix} = \frac{\mathbf{C}\_{inj} \times V\_{inj}}{BV} \tag{11}$$

where *Cinj* is the injected substrate concentration, *Vinj* is the injected volume and *BV* is the bed volume of the IMER.

To determine the inhibition constant (*Ki*) for a test drug a set of inhibitor injections in several different concentrations (*[I]*) at two or more concentration of a substrate should be performed. As noted by Dixon in 1953, if 1/*V* is plotted against inhibitor concentration *[I]*, a straight line plot is obtained for each substrate concentration *[S]*. The *[I]* value of a intersect of those lines is equal to -*Ki*. If curves obtained for several different *[S]* converge in the left upper quadrant of a chart, the inhibitor is competitive. If curves converge on the *[I]* axis, the inhibitor is non-competitive. For not competitive inhibition, the lines are parallel.

Simultaneous injections of both a substrate at a xed saturating concentration and increasing concentration of an inhibitor, result in increasing reduction of the peak area (*Ai*) in comparison to area obtained for a substrate alone (*A0*). The percent inhibition (100 - (*Ai*/*A0* x 100)) is then plotted against the inhibitor concentration to obtain the inhibition curves (Girelli & Mattei, 2005; Nie & Wang, 2009). Recently this technique has been used for the kinetic characterization of inhibitors specific to brain-targeted butyrylcholinesterase (BuChE) (Bartolini et al., 2009), acetylocholinesterase (AchE) (Bartolini et al., 2004; Bartolini et al., 2005; He et al., 2010) and βsecretase (human recombinant β-amyloid precursor protein cleaving enzyme, hrBACE1)(Mancini & Andrisano, 2010) as potential therapeutics for Alzheimer's disease. IMER was also used for rapid and cost-effective on-line chromatographic screening of matrix metalloproteinases (MMP-9 (Ma & Chun Yong Chan, 2010) and MMP-8 (Mazzini et al., 2011)) inhibitors that may be useful in cancer therapy and for determining the role of some derived plant products for treating NO-dependent smooth disorders using monolithic micro-IMER with covalently bounded arginase (André et al., 2011).

### **5. Conclusions**

The drive to bring innovative drugs to market faster, without compromising quality and safety, induced need for new experimental techniques and methodologies. It is crucial to

Affinity Chromatography as a Tool for Quantification of

(Print)0003-2700 (Linking)

(Electronic)1860-7179 (Linking)

1087-0156 (Print)1087-0156 (Linking)

1387-2273 (Print)1387-2273 (Linking)

ISSN 0014-2956 (Print)0014-2956 (Linking)

73, ISSN 1570-0232 (Print)1570-0232 (Linking)

pp. 170-171, ISSN 0264-6021 (Print)0264-6021 (Linking)

7193, ISSN 0003-2700 (Print)0003-2700 (Linking)

925, ISSN 1873-264X (Electronic)0731-7085 (Linking)

Interactions Between Drug Molecules and Their Protein Targets 297

Beigi, F., & Wainer, I. W. (2003). Syntheses of immobilized G protein-coupled receptor

Beigi, F., Chakir, K., Xiao, R. P., & Wainer, I. W. (2004). G-protein-coupled receptor

Calleri, E., Temporini, C., & Massolini, G. (2011). Frontal affinity chromatography in

Calleri, E., Temporini, C., Caccialanza, G., & Massolini, G. (2009). Target-based drug

Chan, N. W., Lewis, D. F., Rosner, P. J., Kelly, M. A., & Schriemer, D. C. (2003). Frontal

Chen, J., & Hage, D. S. (2004). Quantitative analysis of allosteric drug-protein binding by

Chen, J., Ohnmacht, C., & Hage, D. S. (2004). Studies of phenytoin binding to human serum

Cheng, Y., Ho, E., Subramanyam, B., & Tseng, J. L. (2004). Measurements of drug-protein

Dixon, M. (1953). The determination of enzyme inhibitor constants. *Biochem J,* Vol. 55, No. 1,

Girelli, A. M., & Mattei, E. (2005). Application of immobilized enzyme reactor in on-line

Gottschalk, I., Li, Y. M., & Lundahl, P. (2000). Chromatography on cells: analyses of solute

Gottschalk, I., Lundqvist, A., Zeng, C. M., Hagglund, C. L., Zuo, S. S., Brekkan, E., Eaker, D.,

319, No. 1, pp. 1-12, ISSN 0003-2697 (Print)0003-2697 (Linking)

chromatographic stationary phases: characterization of immobilized mu and kappa opioid receptors. *Anal Chem,* Vol. 75, No. 17, pp. 4480-4485, ISSN 0003-2700

chromatographic stationary phases. 2. Ligand-induced conformational mobility in an immobilized beta2-adrenergic receptor. *Anal Chem,* Vol. 76, No. 24, pp. 7187-

characterizing immobilized receptors. *J Pharm Biomed Anal,* Vol. 54, No. 5, pp. 911-

discovery: the emerging success of frontal affinity chromatography coupled to mass spectrometry. *ChemMedChem,* Vol. 4, No. 6, pp. 905-916, ISSN 1860-7187

affinity chromatography-mass spectrometry assay technology for multiple stages of drug discovery: applications of a chromatographic biosensor. *Anal Biochem,* Vol.

biointeraction chromatography. *Nat Biotechnol,* Vol. 22, No. 11, pp. 1445-1448, ISSN

albumin by high-performance affinity chromatography. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 809, No. 1, pp. 137-145, ISSN 1570-0232 (Print)1570-0232 (Linking)

binding by using immobilized human serum albumin liquid chromatography-mass spectrometry. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 809, No. 1, pp. 67-

high performance liquid chromatography: a review. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 819, No. 1, pp. 3-16, ISSN 1570-0232 (Print)1570-0232 (Linking)

interactions with the glucose transporter Glut1 in human red cells adsorbed on lectin-gel beads. *J Chromatogr B Biomed Sci Appl,* Vol. 739, No. 1, pp. 55-62, ISSN

& Lundahl, P. (2000). Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter. *Eur J Biochem,* Vol. 267, No. 23, pp. 6875-6882,

determine biological activity, drug-target interactions and physico-chemical properties of drug candidates as predictors of administration, distribution, metabolism, excretion (ADME) characteristics. In fact, development of rapid methodologies enabling to obtain those information is a key aspects of the drug discovery process. QAC as rapid, relatively simple technique with the possibility of automation proved to be useful alternative to conventional methods in the eld of drug discovery and analysis. This method is facilitated by multiple use of the same column with the immobilized target and as a consequence the reproducibility of assays is increased. Thanks to the above features QAC becomes popular method of measuring binding affinity of the drug-protein interactions. Variety of data that we can obtain via this technique allows characterization of binding reactions as well as description of the binding site. The development of techniques of high-yielding synthesis increases demand for technology that would allow pharmaceutical companies for efficient and rapid biological screening of thousands of synthesized compounds (Renaud & Delsuc, 2009). Taking into account that approximately one drug is produced after 8000-10000 compounds were subjected to primary and secondary drug screens, the classical approach, one compound-one assay, becomes unsatisfactory (Caldwell, 2000; as cited in Nie & Wang, 2009) . Studies conducted by Ng and colleagues have shown that use of an automated high performance chromatographic system consisting of two affinity columns and a mass spectrometer as the detector, allow to analyse and rank activity of 10 000 compounds in just 24 hours (Ng et al., 2007). Thus QAC techniques seem to be an promising method of preliminary verification of drug candidates, which is an alternative to expensive and tedious in vitro assays.

### **6. References**

determine biological activity, drug-target interactions and physico-chemical properties of drug candidates as predictors of administration, distribution, metabolism, excretion (ADME) characteristics. In fact, development of rapid methodologies enabling to obtain those information is a key aspects of the drug discovery process. QAC as rapid, relatively simple technique with the possibility of automation proved to be useful alternative to conventional methods in the eld of drug discovery and analysis. This method is facilitated by multiple use of the same column with the immobilized target and as a consequence the reproducibility of assays is increased. Thanks to the above features QAC becomes popular method of measuring binding affinity of the drug-protein interactions. Variety of data that we can obtain via this technique allows characterization of binding reactions as well as description of the binding site. The development of techniques of high-yielding synthesis increases demand for technology that would allow pharmaceutical companies for efficient and rapid biological screening of thousands of synthesized compounds (Renaud & Delsuc, 2009). Taking into account that approximately one drug is produced after 8000-10000 compounds were subjected to primary and secondary drug screens, the classical approach, one compound-one assay, becomes unsatisfactory (Caldwell, 2000; as cited in Nie & Wang, 2009) . Studies conducted by Ng and colleagues have shown that use of an automated high performance chromatographic system consisting of two affinity columns and a mass spectrometer as the detector, allow to analyse and rank activity of 10 000 compounds in just 24 hours (Ng et al., 2007). Thus QAC techniques seem to be an promising method of preliminary verification of drug candidates, which is an alternative to expensive and tedious

Andre, C., Herlem, G., Gharbi, T., & Guillaume, Y. C. (2011). A new arginase enzymatic

Bartolini, M., Cavrini, V., & Andrisano, V. (2004). Monolithic micro-immobilized-enzyme

Bartolini, M., Cavrini, V., & Andrisano, V. (2005). Choosing the right chromatographic

Bartolini, M., Greig, N. H., Yu, Q. S., & Andrisano, V. (2009). Immobilized

Basiaga, S. B., & Hage, D. S. (2010). Chromatographic studies of changes in binding of

reactor: development and application for the research of plant-derived inhibitors. *J Pharm Biomed Anal,* Vol. 55, No. 1, pp. 48-53, ISSN 1873-264X (Electronic)0731-7085

reactor with human recombinant acetylcholinesterase for on-line inhibition studies. *J Chromatogr A,* Vol. 1031, No. 1-2, pp. 27-34, ISSN 0021-9673 (Print)0021-9673 (Linking)

support in making a new acetylcholinesterase-micro-immobilised enzyme reactor for drug discovery. *J Chromatogr A,* Vol. 1065, No. 1, pp. 135-144, ISSN 0021-9673

butyrylcholinesterase in the characterization of new inhibitors that could ease Alzheimer's disease. *J Chromatogr A,* Vol. 1216, No. 13, pp. 2730-2738, ISSN 1873-

sulfonylurea drugs to human serum albumin due to glycation and fatty acids. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 878, No. 30, pp. 3193-3197, ISSN

in vitro assays.

**6. References**

(Linking)

(Print)0021-9673 (Linking)

3778 (Electronic)0021-9673 (Linking)

1873-376X (Electronic)1570-0232 (Linking)

Affinity Chromatography as a Tool for Quantification of

169-174, ISSN 1570-0232 (Print)1570-0232 (Linking)

4021, ISSN 0022-2623 (Print)0022-2623 (Linking)

ISSN 1570-0232 (Print)1570-0232 (Linking)

110, ISSN 0829-8211 (Print)0829-8211 (Linking)

ISSN 0003-2700 (Print)0003-2700 (Linking)

978-0-8247-4057-3, Boca Raton

3981 (Linking)

(Print)0021-9673 (Linking)

1?, pp. 261-264, ISSN 0021-924X (Print)0021-924X (Linking)

Interactions Between Drug Molecules and Their Protein Targets 299

Jozwiak, K., Moaddel, R., Yamaguchi, R., Ravichandran, S., Collins, J. R., & Wainer, I. W.

Jozwiak, K., Ravichandran, S., Collins, J. R., & Wainer, I. W. (2004). Interaction of

Kasai, K., & Ishii, S. (1975). Quantitative analysis of affinity chromatography of trypsin. A

Kim, H. S., & Wainer, I. W. (2008). Rapid analysis of the interactions between drugs and

Kim, H.S., Hage, D.S., Immobilization methods for affinity chromatography, In: *Handbook of*

Kimura, T., Perry, J., Anzai, N., Pritchard, J. B., & Moaddel, R. (2007). Development and

Kitabatake, T., Moaddel, R., Cole, R., Gandhari, M., Frazier, C., Hartenstein, J., Rosenberg,

Lebert, J. M., Forsberg, E. M., & Brennan, J. D. (2008). Solid-phase assays for small molecule

Loun, B., & Hage, D. S. (1992). Characterization of thyroxine-albumin binding using high-

Loun, B., & Hage, D. S. (1994). Chiral separation mechanisms in protein-based HPLC

No. 22, pp. 8673-8680, ISSN 1520-6882 (Electronic)0003-2700 (Linking) Landry, Y., & Gies, J. P. (2008). Drugs and their molecular targets: an updated overview.

(2005). Qualitative assessment of IC50 values of inhibitors of the neuronal nicotinic acetylcholine receptor using a single chromatographic experiment and multivariate cluster analysis. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 819, No. 1, pp.

noncompetitive inhibitors with an immobilized alpha3beta4 nicotinic acetylcholine receptor investigated by affinity chromatography, quantitative-structure activity relationship analysis, and molecular docking. *J Med Chem,* Vol. 47, No. 16, pp. 4008-

new technique for investigation of protein-ligand interaction. *J Biochem,* Vol. 77, No.

human serum albumin (HSA) using high-performance affinity chromatography (HPAC). *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 870, No. 1, pp. 22-26,

*Affinity Chromatography,* Hage, D.S., pp. 35-79, CRC Press/Taylor&Francis, ISBN

characterization of immobilized human organic anion transporter-based liquid chromatographic stationary phase: hOAT1 and hOAT2. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 859, No. 2, pp. 267-271, ISSN 1570-0232 (Print)1570-0232 (Linking)

A., Bernier, M., & Wainer, I. W. (2008). Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines. *Anal Chem,* Vol. 80,

*Fundam Clin Pharmacol,* Vol. 22, No. 1, pp. 1-18, ISSN 1472-8206 (Electronic)0767-

screening using sol-gel entrapped proteins. *Biochem Cell Biol,* Vol. 86, No. 2, pp. 100-

performance affinity chromatography. I. Interactions at the warfarin and indole sites of albumin. *J Chromatogr,* Vol. 579, No. 2, pp. 225-235, ISSN 0021-9673

columns. 1. Thermodynamic studies of (R)- and (S)-warfarin binding to immobilized human serum albumin. *Anal Chem,* Vol. 66, No. 21, pp. 3814-3822,

Gritti, F., & Guiochon, G. (2005). Critical contribution of nonlinear chromatography to the

Guiffant, D., Tribouillard, D., Gug, F., Galons, H., Meijer, L., Blondel, M., & Bach, S. (2007).

Hage, D. S. (2002). High-performance affinity chromatography: a powerful tool for studying

Hage, D. S., Jackson, A., Sobansky, M. R., Schiel, J. E., Yoo, M. J., & Joseph, K. S. (2009).

Hage, D. S., Ruhn, P. F. (2006). An Introduction to Affinity Chromatography, In: *Handbook of*

Hage, D.S. (Ed.). (2006). *Handbook of Affinity Chromatography,* CRC Press/Taylor&Francis,

Hage, D.S., Chen, J. (2006). Quantitative Affinity Chromatography: Practical Aspect, In:

He, P., Davies, J., Greenway, G., & Haswell, S. J. (2010). Measurement of acetylcholinesterase

Joseph, K. S., & Hage, D. S. (2010). Characterization of the binding of sulfonylurea drugs to

Josic, D., & Buchacher, A. (2001). Application of monoliths as supports for affinity

Jozwiak, K., Haginaka, J., Moaddel, R., & Wainer, I. W. (2002). Displacement and nonlinear

Jozwiak, K., Hernandez, S. C., Kellar, K. J., & Wainer, I. W. (2003). Enantioselective

No. 1-3, pp. 153-174, ISSN 0165-022X (Print)0165-022X (Linking)

Press/Taylor&Francis, ISBN 978-0-8247-4057-3, Boca Raton

ISSN 1860-7314 (Electronic)1860-6768 (Linking)

(Electronic)1615-9306 (Linking)

978-0-8247-4057-3, Boca Raton

ISBN 978-0-8247-4057-3, Boca Raton, FL

4324 (Electronic)0003-2670 (Linking)

0003-2700 (Print)0003-2700 (Linking)

ISSN 1570-0232 (Print)1570-0232 (Linking)

0232 (Linking)

pp. 3-30, ISSN 1570-0232 (Print)1570-0232 (Linking)

understanding of retention mechanism in reversed-phase liquid chromatography. *J Chromatogr A,* Vol. 1099, No. 1-2, pp. 1-42, ISSN 0021-9673 (Print)0021-9673 (Linking)

Identification of intracellular targets of small molecular weight chemical compounds using affinity chromatography. *Biotechnol J,* Vol. 2, No. 1, pp. 68-75,

serum protein binding. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 768, No. 1,

Characterization of drug-protein interactions in blood using high-performance affinity chromatography. *J Sep Sci,* Vol. 32, No. 5-6, pp. 835-853, ISSN 1615-9314

*Affinity Chromatography,* Hage, D.S., pp. 3-15, CRC Press/Taylor&Francis, ISBN

*Handbook of Affinity Chromatography,* Hage, D.S., pp. 595-628, CRC

inhibition using bienzymes immobilized monolith micro-reactor with integrated electrochemical detection. *Anal Chim Acta,* Vol. 659, No. 1-2, pp. 9-14, ISSN 1873-

HSA by high-performance affinity chromatography. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 878, No. 19, pp. 1590-1598, ISSN 1873-376X (Electronic)1570-

chromatography and fast enzymatic conversion. *J Biochem Biophys Methods,* Vol. 49,

chromatographic techniques in the investigation of interaction of noncompetitive inhibitors with an immobilized alpha3beta4 nicotinic acetylcholine receptor liquid chromatographic stationary phase. *Anal Chem,* Vol. 74, No. 18, pp. 4618-4624, ISSN

interactions of dextromethorphan and levomethorphan with the alpha 3 beta 4 nicotinic acetylcholine receptor: comparison of chromatographic and functional data. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 797, No. 1-2, pp. 373-379,

Affinity Chromatography as a Tool for Quantification of

(Print)0003-2700 (Linking)

0003-2700 (Print)0003-2700 (Linking)

2697 (Linking)

(Print)1570-0232 (Linking)

(Print)1087-0571 (Linking)

Interactions Between Drug Molecules and Their Protein Targets 301

Moaddel, R., Cloix, J. F., Ertem, G., & Wainer, I. W. (2002). Multiple receptor liquid

*Res,* Vol. 19, No. 1, pp. 104-107, ISSN 0724-8741 (Print)0724-8741 (Linking) Moaddel, R., Jozwiak, K., Whittington, K., & Wainer, I. W. (2005). Conformational mobility

Moaddel, R., Lu, L., Baynham, M., & Wainer, I. W. (2002). Immobilized receptor- and

Moaddel, R., Oliveira, R. V., Kimura, T., Hyppolite, P., Juhaszova, M., Xiao, Y., Kellar, K. J.,

Moaddel, R., Rosenberg, A., Spelman, K., Frazier, J., Frazier, C., Nocerino, S., Brizzi, A.,

Moaddel, R., Yamaguchi, R., Ho, P. C., Patel, S., Hsu, C. P., Subrahmanyam, V., & Wainer, I.

Narayanan, S. R., Kakodkar, S. V., & Crane, L. J. (1990). "Glutaraldehyde-P", a stable,

Ng, W., Dai, J. R., Slon-Usakiewicz, J. J., Redden, P. R., Pasternak, A., & Reid, N. (2007).

Nie, Y.-l., Wang, W.-h. (2009) .Immobilized Enzyme Reactor in On-line LC and Its Application in Drug Screening. *Chromatographia,* Vol. 69, pp. S5–S12

pp. 278-284, ISSN 0003-2697 (Print)0003-2697 (Linking)

Vol. 768, No. 1, pp. 41-53, ISSN 1570-0232 (Print)1570-0232 (Linking) Moaddel, R., Musyimi, H. K., Sanghvi, M., Bashore, C., Frazier, C. R., Khadeer, M., Bhatia,

pp. 416-419, ISSN 1873-264X (Electronic)0731-7085 (Linking)

2, pp. 216-218, ISSN 0003-2697 (Print)0003-2697 (Linking)

chromatography stationary phase for online screening. *Anal Biochem,* Vol. 364, No.

chromatographic stationary phases: the co-immobilization of nicotinic receptors, gamma-amino-butyric acid receptors, and N-methyl D-aspartate receptors. *Pharm*

of immobilized alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nicotinic acetylcholine receptors. *Anal Chem,* Vol. 77, No. 3, pp. 895-901, ISSN 0003-2700

transporter-based liquid chromatographic phases for on-line pharmacological and biochemical studies: a mini-review. *J Chromatogr B Analyt Technol Biomed Life Sci,*

P., & Wainer, I. W. (2010). Synthesis and characterization of a cellular membrane affinity chromatography column containing histamine 1 and P2Y(1) receptors: a multiple G-protein coupled receptor column. *J Pharm Biomed Anal,* Vol. 52, No. 3,

Bernier, M., & Wainer, I. W. (2008). Initial synthesis and characterization of an alpha7 nicotinic receptor cellular membrane affinity chromatography column: effect of receptor subtype and cell type. *Anal Chem,* Vol. 80, No. 1, pp. 48-54, ISSN

Mugnaini, C., & Wainer, I. W. (2011). Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening. *Anal Biochem,* Vol. 412, No. 1, pp. 85-91, ISSN 1096-0309 (Electronic)0003-

W. (2005). Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 818, No. 2, pp. 263-268, ISSN 1570-0232

reactive aldehyde matrix for affinity chromatography. *Anal Biochem,* Vol. 188, No. 2,

Automated multiple ligand screening by frontal affinity chromatography-mass spectrometry (FAC-MS). *J Biomol Screen,* Vol. 12, No. 2, pp. 167-174, ISSN 1087-0571

Loun, B., & Hage, D. S. (1995). Characterization of thyroxine-albumin binding using high-

Lundqvist, A., Ocklind, G., Haneskog, L., & Lundahl, P. (1998). Freeze-thaw immobilization

Ma, X., & Chan, E. C. (2010). On-line chromatographic screening of matrix metalloproteinase

Mallik, R., Yoo, M. J., Chen, S., & Hage, D. S. (2008). Studies of verapamil binding to human

Mancini, F., & Andrisano, V. (2010). Development of a liquid chromatographic system with

Markuszewski, M., & Kaliszan, R. (2002). Quantitative structure-retention relationships in

Mateo, C., Fernandez-Lorente, G., Abian, O., Fernandez-Lafuente, R., & Guisan, J. M. (2000).

Mazzini, F., Nuti, E., Petri, A., & Rossello, A. (2011). Immobilization of matrix

Moaddel, R., & Wainer, I. W. (2006). Development of immobilized membrane-based affinity

Moaddel, R., Bullock, P. L., & Wainer, I. W. (2004). Development and characterization of an

*Sci,* Vol. 799, No. 2, pp. 255-263, ISSN 1570-0232 (Print)1570-0232 (Linking) Moaddel, R., Calleri, E., Massolini, G., Frazier, C. R., & Wainer, I. W. (2007). The synthesis

ISSN 1572-6495 (Print)1572-6495 (Linking)

(Print)0952-3499 (Linking)

(Electronic)0731-7085 (Linking)

1525-7797 (Print)1525-7797 (Linking)

(Electronic)1570-0232 (Linking)

4324 (Electronic)0003-2670 (Linking)

0232 (Linking)

0232 (Linking)

performance affinity chromatography. II. Comparison of the binding of thyroxine, triiodothyronines and related compounds at the warfarin and indole sites of human serum albumin. *J Chromatogr B Biomed Appl,* Vol. 665, No. 2, pp. 303-314,

of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate. *J Mol Recognit,* Vol. 11, No. 1-6, pp. 52-57, ISSN 0952-3499

inhibitors using immobilized MMP-9 enzyme reactor. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 878, No. 21, pp. 1777-1783, ISSN 1873-376X (Electronic)1570-

serum albumin by high-performance affinity chromatography. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 876, No. 1, pp. 69-75, ISSN 1570-0232 (Print)1570-

fluorescent detection for beta-secretase immobilized enzyme reactor on-line enzymatic studies. *J Pharm Biomed Anal,* Vol. 52, No. 3, pp. 355-361, ISSN 1873-264X

affinity high-performance liquid chromatography. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 768, No. 1, pp. 55-66, ISSN 1570-0232 (Print)1570-0232 (Linking)

Multifunctional epoxy supports: a new tool to improve the covalent immobilization of proteins. The promotion of physical adsorptions of proteins on the supports before their covalent linkage. *Biomacromolecules,* Vol. 1, No. 4, pp. 739-745, ISSN

metalloproteinase 8 (MMP-8) for online drug screening. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 879, No. 11-12, pp. 756-762, ISSN 1873-376X

columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations. *Anal Chim Acta,* Vol. 564, No. 1, pp. 97-105, ISSN 1873-

open tubular column containing immobilized P-glycoprotein for rapid on-line screening for P-glycoprotein substrates. *J Chromatogr B Analyt Technol Biomed Life*

and initial characterization of an immobilized purinergic receptor (P2Y1) liquid

chromatography stationary phase for online screening. *Anal Biochem,* Vol. 364, No. 2, pp. 216-218, ISSN 0003-2697 (Print)0003-2697 (Linking)

Affinity Chromatography as a Tool for Quantification of

Vol. 59, No. 9, pp. 1286–1295

2273 (Linking)

(Linking)

(Linking)

(Electronic)1570-0232 (Linking)

1671-4083 (Print)1671-4083 (Linking)

91-98, ISSN 0006-3002 (Print)0006-3002 (Linking)

ISSN 1387-2273 (Print)1387-2273 (Linking)

Interactions Between Drug Molecules and Their Protein Targets 303

Wade, J.L., Bergold, A.F., Carr, P.W. (1987). Theoretical description of nonlinear

Wainer, I. W., Zhang, Y., Xiao, Y., & Kellar, K. J. (1999). Liquid chromatographic studies

Winzor, D. J. (2004). Determination of binding constants by affinity chromatography. *J*

Xuan, H., & Hage, D. S. (2005). Immobilization of alpha(1)-acid glycoprotein for

Yang, J., & Hage, D. S. (1993). Characterization of the binding and chiral separation of D- and

Yoo, M. J., Smith, Q. R., & Hage, D. S. (2009). Studies of imipramine binding to human

Yu, W., Yuan, B., Deng, X., He, L., Youyi, Z., & Qide, H. (2005). The preparation of HEK293

Zeng, C. M., Zhang, Y., Lu, L., Brekkan, E., Lundqvist, A., & Lundahl, P. (1997).

Zhang, Y., Leonessa, F., Clarke, R., & Wainer, I. W. (2000). Development of an immobilized

Vol. 339, No. 2, pp. 198-205, ISSN 0003-2697 (Print)0003-2697 (Linking) Yuan, B. X., Hou, J., He, L. C., & Yang, G. D. (2005). Evaluation of drug-muscarinic receptor

300-310, ISSN 0003-2697 (Print)0003-2697 (Linking)

chromatography, with applications to physicochemical measurements in affinity chromatography and implications for preparative-scale separations. *Anal. Chem.*,

with immobilized neuronal nicotinic acetylcholine receptor stationary phases: effects of receptor subtypes, pH and ionic strength on drug-receptor interactions. *J Chromatogr B Biomed Sci Appl,* Vol. 724, No. 1, pp. 65-72, ISSN 1387-2273 (Print)1387-

*Chromatogr A,* Vol. 1037, No. 1-2, pp. 351-367, ISSN 0021-9673 (Print)0021-9673

chromatographic studies of drug-protein binding. *Anal Biochem,* Vol. 346, No. 2, pp.

L-tryptophan on a high-performance immobilized human serum albumin column. *J Chromatogr,* Vol. 645, No. 2, pp. 241-250, ISSN 0021-9673 (Print)0021-9673 (Linking) Yang, Q., & Lundahl, P. (1995). Immobilized proteoliposome affinity chromatography for

quantitative analysis of specific interactions between solutes and membrane proteins. Interaction of cytochalasin B and D-glucose with the glucose transporter Glut1. *Biochemistry,* Vol. 34, No. 22, pp. 7289-7294, ISSN 0006-2960 (Print)0006-2960

serum albumin by high-performance affinity chromatography. *J Chromatogr B Analyt Technol Biomed Life Sci,* Vol. 877, No. 11-12, pp. 1149-1154, ISSN 1873-376X

alpha1A or HEK293 alpha1B cell membrane stationary phase and the chromatographic affinity study of ligands of alpha1 adrenoceptor. *Anal Biochem,*

affinities using cell membrane chromatography and radioligand binding assay in guinea pig jejunum membrane. *Acta Pharmacol Sin,* Vol. 26, No. 1, pp. 113-116, ISSN

Immobilization of human red cells in gel particles for chromatographic activity studies of the glucose transporter Glut1. *Biochim Biophys Acta,* Vol. 1325, No. 1, pp.

P-glycoprotein stationary phase for on-line liquid chromatographic determination of drug-binding affinities. *J Chromatogr B Biomed Sci Appl,* Vol. 739, No. 1, pp. 33-37,

Pidgeon, C., & Venkataram, U. V. (1989). Immobilized artificial membrane chromatography:

Renaud, J. P., & Delsuc, M. A. (2009). Biophysical techniques for ligand screening and drug

Rizzi, L., Dallanoce, C., Matera, C., Magrone, P., Pucci, L., Gotti, C., Clementi, F., & De

Ruhn, P. F., Garver, S., & Hage, D. S. (1994). Development of dihydrazide-activated silica

Sanghvi, M., Moaddel, R., Frazier, C., & Wainer, I. W. (2010). Synthesis and characterization

Slon-Usakiewicz, J. J., Ng, W., Dai, J. R., Pasternak, A., & Redden, P. R. (2005). Frontal

Soczewinski, E., Bieganowska, M. (1969). Investigations on the relationship between

Taylor, R.F. (Ed.). (1991). *Protein Immobilization: Fundamentals and Applications*, Marcel

Temporini, C., Ceruti, S., Calleri, E., Ferrario, S., Moaddel, R., Abbracchio, M. P., &

Trujillo, C. A., Majumder, P., Gonzalez, F. A., Moaddel, R., & Ulrich, H. (2007). Immobilized

Turkova, J. (1999). Oriented immobilization of biologically active proteins as a tool for

Tweed, S. A., Loun, B., & Hage, D. S. (1997). Effects of ligand heterogeneity in the

722, No. 1-2, pp. 11-31, ISSN 1387-2273 (Print)1387-2273 (Linking)

No. 1-2, pp. 9-19, ISSN 0021-9673 (Print)0021-9673 (Linking)

chromatography. *J Chromatogr A*, Vol. 40, pp. 431-439

pp. 701-710, ISSN 0731-7085 (Print)0731-7085 (Linking)

pp. 4790-4798, ISSN 0003-2700 (Print)0003-2700 (Linking)

Dekker, ISBN10<PHONE_NUMBER>, New York

(Electronic)0003-2697 (Linking)

ISSN 0003-2697 (Print)0003-2697 (Linking)

(Electronic)1471-4892 (Linking)

3405 (Electronic)0960-894X (Linking)

(Electronic)0731-7085 (Linking)

supports composed of membrane lipids. *Anal Biochem,* Vol. 176, No. 1, pp. 36-47,

design. *Curr Opin Pharmacol,* Vol. 9, No. 5, pp. 622-628, ISSN 1471-4973

Amici, M. (2008). Epiboxidine and novel-related analogues: a convenient synthetic approach and estimation of their affinity at neuronal nicotinic acetylcholine receptor subtypes. *Bioorg Med Chem Lett,* Vol. 18, No. 16, pp. 4651-4654, ISSN 1464-

supports for high-performance affinity chromatography. *J Chromatogr A,* Vol. 669,

of liquid chromatographic columns containing the immobilized ligand binding domain of the estrogen related receptor alpha and estrogen related receptor gamma. *J Pharm Biomed Anal,* Vol. 53, No. 3, pp. 777-780, ISSN 1873-264X

affinity chromatography with MS detection (FAC-MS) in drug discovery. *Drug Discov Today,* Vol. 10, No. 6, pp. 409-416, ISSN 1359-6446 (Print)1359-6446 (Linking)

molecular structure and chromatogenic parameters: I. Two homologous series of pyridine derivatives suitable as reference compounds in liquid-liquid partition

Massolini, G. (2009). Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry. *Anal Biochem,* Vol. 384, No. 1, pp. 123-129, ISSN 1096-0309

P2X2 purinergic receptor stationary phase for chromatographic determination of pharmacological properties and drug screening. *J Pharm Biomed Anal,* Vol. 44, No. 3,

revealing protein interactions and function. *J Chromatogr B Biomed Sci Appl,* Vol.

characterization of affinity columns by frontal analysis. *Anal Chem,* Vol. 69, No. 23,

**Part 9**

**Practical Application**

**of Affinity Chromatography in Research Field**

## **Part 9**

**Practical Application of Affinity Chromatography in Research Field**

304 Affinity Chromatography

Zhang, Y., Xiao, Y., Kellar, K. J., & Wainer, I. W. (1998). Immobilized nicotinic receptor

Zhu, L., Chen, L., Luo, H., & Xu, X. (2003). Frontal affinity chromatography combined on-

(Linking)

0003-2700 (Print)0003-2700 (Linking)

stationary phase for on-line liquid chromatographic determination of drug-receptor affinities. *Anal Biochem,* Vol. 264, No. 1, pp. 22-25, ISSN 0003-2697 (Print)0003-2697

line with mass spectrometry: a tool for the binding study of different epidermal growth factor receptor inhibitors. *Anal Chem,* Vol. 75, No. 23, pp. 6388-6393, ISSN

**15**

*<sup>2</sup>BIA Separations,*

*1,2,3Slovenia*

**The Value of Fungal Protease Inhibitors**

Matjaž Peterka2,3, Miloš Barut2,3, Janko Kos1 and Jože Brzin<sup>1</sup>

*<sup>3</sup>Centre of Excellence for Biosensors, Instrumentation and Process Control,*

Proteolytic enzymes (also known as proteases, proteinases or peptidases) offer a wide range of applications. They are routinely used in detergent, leather, food and pharmaceutical industries, as well as in medical and basic research. Therefore, effective isolation procedures are of great importance. The chapter describes the use of recently discovered protease inhibitors from basidiomycetes as affinity chromatography ligands for isolating proteases. Affinity columns with serine and cysteine protease inhibitors immobilized to the natural polymer Sepharose have been prepared, the chromatography procedure optimized and used for isolating proteases from various bacterial, plant and animal sources. The cysteine protease inhibitor macrocypin showed superior characteristics as a ligand, so was selected for immobilization to CIM (Convective Interaction Media) monolithic disks. Different immobilization chemistries and process conditions were optimized to determine the best conditions for high capacity and selectivity. A very effective method for isolating cysteine proteases was developed using affinity chromatography with the fungal cysteine protease

Proteases occur in all groups of organisms, including bacteria, archaea, protists, fungi, plants, animals and viruses. They are classified according to their catalytic type into aspartic, cysteine, glutamic, serine and threonine peptidases, based on the type of the amino acid residue at the active site, and metallopeptidases, that require a catalytic divalent metal ion within the active site. The MEROPS database (http://merops.sanger.ac.uk) further classifies peptidases according to their evolutionary relationships and encompasses information on all

Proteases offer a wide range of biotechnological applications. Alkaline proteases are routinely used in the detergent industry. Proteases with elastolytic and keratinolytic activities have been used in the leather industry for dehairing and baiting skins and hides. They are used in the food industry in cheese making and baking, in preparing the various

inhibitor macrocypin immobilized to a CIM monolithic disk.

**1.1 Proteases and their applications**

known proteases (Rawlings et al., 2010).

**1. Introduction**

 **in Affinity Chromatography**

*<sup>1</sup>Department of Biotechnology, Jožef Stefan Institute,*

Jerica Sabotič1, Katarina Koruza1,2, Boštjan Gabor2,3,

## **The Value of Fungal Protease Inhibitors in Affinity Chromatography**

Jerica Sabotič1, Katarina Koruza1,2, Boštjan Gabor2,3, Matjaž Peterka2,3, Miloš Barut2,3, Janko Kos1 and Jože Brzin<sup>1</sup> *<sup>1</sup>Department of Biotechnology, Jožef Stefan Institute, <sup>2</sup>BIA Separations, <sup>3</sup>Centre of Excellence for Biosensors, Instrumentation and Process Control, 1,2,3Slovenia*

### **1. Introduction**

Proteolytic enzymes (also known as proteases, proteinases or peptidases) offer a wide range of applications. They are routinely used in detergent, leather, food and pharmaceutical industries, as well as in medical and basic research. Therefore, effective isolation procedures are of great importance. The chapter describes the use of recently discovered protease inhibitors from basidiomycetes as affinity chromatography ligands for isolating proteases. Affinity columns with serine and cysteine protease inhibitors immobilized to the natural polymer Sepharose have been prepared, the chromatography procedure optimized and used for isolating proteases from various bacterial, plant and animal sources. The cysteine protease inhibitor macrocypin showed superior characteristics as a ligand, so was selected for immobilization to CIM (Convective Interaction Media) monolithic disks. Different immobilization chemistries and process conditions were optimized to determine the best conditions for high capacity and selectivity. A very effective method for isolating cysteine proteases was developed using affinity chromatography with the fungal cysteine protease inhibitor macrocypin immobilized to a CIM monolithic disk.

### **1.1 Proteases and their applications**

Proteases occur in all groups of organisms, including bacteria, archaea, protists, fungi, plants, animals and viruses. They are classified according to their catalytic type into aspartic, cysteine, glutamic, serine and threonine peptidases, based on the type of the amino acid residue at the active site, and metallopeptidases, that require a catalytic divalent metal ion within the active site. The MEROPS database (http://merops.sanger.ac.uk) further classifies peptidases according to their evolutionary relationships and encompasses information on all known proteases (Rawlings et al., 2010).

Proteases offer a wide range of biotechnological applications. Alkaline proteases are routinely used in the detergent industry. Proteases with elastolytic and keratinolytic activities have been used in the leather industry for dehairing and baiting skins and hides. They are used in the food industry in cheese making and baking, in preparing the various

The Value of Fungal Protease Inhibitors in Affinity Chromatography 309

cysteine protease inhibitor family (I79) with only one representative found in one plant

The serine protease inhibitor cnispin (family I66), identified in the mushroom *Clitocybe nebularis*, is a 16.4 kDa protein with acidic isoelectric point. It is a very stable protease inhibitor that resists short-term exposure to extremes of pH (between pH 2 and pH 11). It is a very strong inhibitor of trypsin (family S1) with *K*i in the nanomolar range, and a weak inhibitor of chymotrypsin (family S1), with *K*i in the micromolar range. Inhibition of kallikrein (family S1) and subtilisin (family S8) is very weak and other proteases are not

Mycocypins are cysteine protease inhibitors unique to basidiomycete mushrooms and belong to two MEROPS families (I48 and I85). Family I48 is represented by clitocypin, identified in *Clitocybe nebularis* (Brzin et al., 2000; Renko et al., 2010; Sabotič et al., 2006) and family I85 by macrocypins identified from *Macrolepiota procera* (Renko et al., 2010; Sabotič et al., 2009b). These are small (16.8 to 20 kDa) and exceptionally stable proteins, exhibiting high thermal and broad pH stability (Galeša et al., 2004; Kidrič et al., 2002; Sabotič et al., 2009b). They have the β-trefoil fold, which is composed of a core six-stranded β-barrel surrounded by 11 loops (Fig. 1) that provide a versatile surface for the inhibition of several types of proteases (Renko et al., 2010). They are very strong inhibitors of papain-like peptidases (family C1), including papain, cathepsins L, V, S, and K in the low nanomolar range and cathepsins B and H with higher inhibition constants. A second inhibitory reactive site is involved in the inhibition of the cysteine protease asparaginyl endopeptidase (AEP, legumain), of family C13 by clitocypin, macrocypins 1 and 3, and in the inhibition of the serine protease trypsin (family S1) by macrocypin 4 (Renko et al., 2010; Sabotič et al., 2007a;

Fig. 1. Three-dimensional structure of macrocypin 1. The two loops involved in inhibiting papain-like proteases (family C1) are indicated with #, and the loop involved in legumain

pathogenic fungal species (Rawlings, 2010).

inhibited (Avanzo et al., 2009).

Sabotič et al., 2009b).

(family C13) inhibition with \*.

protein hydrolysates used as flavour enhancers, in meat tenderization and in manufacturing protein-rich diets. In the pharmaceutical industry proteases have found uses as therapeutic agents as well as additives in preparations of slow-release dosage forms. In addition to industrial and medical applications, proteases have found use also in basic research, for example proteases with very selective peptide bond cleavage are used in protein sequencing and proteome analyses. Furthermore, unselective proteases are also used, for example proteinase K is used in nucleic acid isolation, and trypsin is widely used in animal cell culture maintenance (Kumar & Takagi, 1999; Østergaard & Olsen, 2010; Rao et al., 1998).

### **1.2 Protease inhibitors**

Proteases play essential metabolic and regulatory functions in many biological processes, therefore the regulation of their activity is essential. Interaction with protease inhibitors constitutes a very important mechanism of protease regulation (Lopez-Otin & Bond, 2008; Rawlings et al., 2010). Protease inhibitors are either small molecules or proteins. They can be classified according to the source organism (microbial, fungal, plant, animal), according to their structure (primary and three-dimensional), or according to their inhibitory profile (broadrange, specific) and reaction mechanism (competitive, non-competitive, uncompetitive as well as reversible or irreversible). Commonly they are classified according to the class of protease they inhibit (for example: aspartic, cysteine or serine protease inhibitors) but a detailed classification of protein protease inhibitors based on their evolutionary relationship is available in the MEROPS database, together with a list of small molecule peptidase inhibitors (http://merops.sanger.ac.uk/inhibitors/). There are two general mechanisms by which protein inhibitors inhibit peptidases - irreversible "trapping" reaction, involving a conformational change of the inhibitor, and reversible tight-binding reactions, where the inhibitor forms a high-affinity interaction with the peptidase, most often at the active site (Christeller, 2005; Rawlings, 2010; Rawlings et al., 2004). The reversible protease inhibitors are used as ligands in affinity chromatography (Cuatrecasas et al., 1968).

### **1.2.1 Protease inhibitors of fungal origin**

The only routinely used small molecule inhibitor of fungal origin is the irreversible inhibitor of cysteine proteases E-64, first isolated from the filamentous fungus *Aspergillus japonicus* (Hanada et al., 1978). Several other small molecule inhibitors with a broad inhibitory spectrum that are routinely used (e.g. pepstatin A, chymostatin, leupeptin, antipain, phosphoramidon, bestatin) were originally isolated from bacteria (Rawlings, 2010).

Information on protein protease inhibitors of fungal origin is limited. No protein metalloprotease inhibitors has been described, and only one family of aspartic protease inhibitors (family I34), which includes the highly specific inhibitor of the yeast proteinase A or saccharopepsin (Phylip et al., 2001). There are four families of serine protease inhibitors, namely the inhibitors of serine carboxypeptidase Y (family I51), *Aspergillus* elastase inhibitor family (I78), inhibitors of subtilisin-like proteases homologous to the subtilisin propeptide (family I9) and trypsin-specific protease inhibitors (family I66). Representatives of the latter two have been identified from higher fungi or mushrooms as well as from filamentous fungi (Rawlings, 2010). Furthermore, only three families of cysteine protease inhibitors have been described, namely mycocypins (families I48 and I85), that are unique to higher fungi, and a

protein hydrolysates used as flavour enhancers, in meat tenderization and in manufacturing protein-rich diets. In the pharmaceutical industry proteases have found uses as therapeutic agents as well as additives in preparations of slow-release dosage forms. In addition to industrial and medical applications, proteases have found use also in basic research, for example proteases with very selective peptide bond cleavage are used in protein sequencing and proteome analyses. Furthermore, unselective proteases are also used, for example proteinase K is used in nucleic acid isolation, and trypsin is widely used in animal cell culture maintenance (Kumar & Takagi, 1999; Østergaard & Olsen, 2010; Rao et al., 1998).

Proteases play essential metabolic and regulatory functions in many biological processes, therefore the regulation of their activity is essential. Interaction with protease inhibitors constitutes a very important mechanism of protease regulation (Lopez-Otin & Bond, 2008; Rawlings et al., 2010). Protease inhibitors are either small molecules or proteins. They can be classified according to the source organism (microbial, fungal, plant, animal), according to their structure (primary and three-dimensional), or according to their inhibitory profile (broadrange, specific) and reaction mechanism (competitive, non-competitive, uncompetitive as well as reversible or irreversible). Commonly they are classified according to the class of protease they inhibit (for example: aspartic, cysteine or serine protease inhibitors) but a detailed classification of protein protease inhibitors based on their evolutionary relationship is available in the MEROPS database, together with a list of small molecule peptidase inhibitors (http://merops.sanger.ac.uk/inhibitors/). There are two general mechanisms by which protein inhibitors inhibit peptidases - irreversible "trapping" reaction, involving a conformational change of the inhibitor, and reversible tight-binding reactions, where the inhibitor forms a high-affinity interaction with the peptidase, most often at the active site (Christeller, 2005; Rawlings, 2010; Rawlings et al., 2004). The reversible protease inhibitors are

The only routinely used small molecule inhibitor of fungal origin is the irreversible inhibitor of cysteine proteases E-64, first isolated from the filamentous fungus *Aspergillus japonicus* (Hanada et al., 1978). Several other small molecule inhibitors with a broad inhibitory spectrum that are routinely used (e.g. pepstatin A, chymostatin, leupeptin, antipain,

Information on protein protease inhibitors of fungal origin is limited. No protein metalloprotease inhibitors has been described, and only one family of aspartic protease inhibitors (family I34), which includes the highly specific inhibitor of the yeast proteinase A or saccharopepsin (Phylip et al., 2001). There are four families of serine protease inhibitors, namely the inhibitors of serine carboxypeptidase Y (family I51), *Aspergillus* elastase inhibitor family (I78), inhibitors of subtilisin-like proteases homologous to the subtilisin propeptide (family I9) and trypsin-specific protease inhibitors (family I66). Representatives of the latter two have been identified from higher fungi or mushrooms as well as from filamentous fungi (Rawlings, 2010). Furthermore, only three families of cysteine protease inhibitors have been described, namely mycocypins (families I48 and I85), that are unique to higher fungi, and a

phosphoramidon, bestatin) were originally isolated from bacteria (Rawlings, 2010).

used as ligands in affinity chromatography (Cuatrecasas et al., 1968).

**1.2.1 Protease inhibitors of fungal origin**

**1.2 Protease inhibitors**

cysteine protease inhibitor family (I79) with only one representative found in one plant pathogenic fungal species (Rawlings, 2010).

The serine protease inhibitor cnispin (family I66), identified in the mushroom *Clitocybe nebularis*, is a 16.4 kDa protein with acidic isoelectric point. It is a very stable protease inhibitor that resists short-term exposure to extremes of pH (between pH 2 and pH 11). It is a very strong inhibitor of trypsin (family S1) with *K*i in the nanomolar range, and a weak inhibitor of chymotrypsin (family S1), with *K*i in the micromolar range. Inhibition of kallikrein (family S1) and subtilisin (family S8) is very weak and other proteases are not inhibited (Avanzo et al., 2009).

Mycocypins are cysteine protease inhibitors unique to basidiomycete mushrooms and belong to two MEROPS families (I48 and I85). Family I48 is represented by clitocypin, identified in *Clitocybe nebularis* (Brzin et al., 2000; Renko et al., 2010; Sabotič et al., 2006) and family I85 by macrocypins identified from *Macrolepiota procera* (Renko et al., 2010; Sabotič et al., 2009b). These are small (16.8 to 20 kDa) and exceptionally stable proteins, exhibiting high thermal and broad pH stability (Galeša et al., 2004; Kidrič et al., 2002; Sabotič et al., 2009b). They have the β-trefoil fold, which is composed of a core six-stranded β-barrel surrounded by 11 loops (Fig. 1) that provide a versatile surface for the inhibition of several types of proteases (Renko et al., 2010). They are very strong inhibitors of papain-like peptidases (family C1), including papain, cathepsins L, V, S, and K in the low nanomolar range and cathepsins B and H with higher inhibition constants. A second inhibitory reactive site is involved in the inhibition of the cysteine protease asparaginyl endopeptidase (AEP, legumain), of family C13 by clitocypin, macrocypins 1 and 3, and in the inhibition of the serine protease trypsin (family S1) by macrocypin 4 (Renko et al., 2010; Sabotič et al., 2007a; Sabotič et al., 2009b).

Fig. 1. Three-dimensional structure of macrocypin 1. The two loops involved in inhibiting papain-like proteases (family C1) are indicated with #, and the loop involved in legumain (family C13) inhibition with \*.

The Value of Fungal Protease Inhibitors in Affinity Chromatography 311

composition of the mobile phase remains unchanged during washing and elution. This technique is known as weak affinity chromatography. When a ligand has strong affinity to the target molecule, it can be released by changing the mobile phase or column conditions. For the most part, elution buffer differs from the application buffer in pH, ionic strength, temperature or polarity. This approach is known as nonspecific elution. Another possibility is biospecific elution, where a buffer with the same pH, ionic strength and polarity as the application buffer is used for elution but with a competing agent that can bind to either the

The role of stationary phase or support in affinity chromatography is ligand immobilization (Hage, 2006). A support is any material to which a biospecific ligand may be covalently attached. Typically, the material to be used as an affinity support is insoluble in the system where the target molecule is found (Hermanson et al., 1992). The support main characteristics are: large specific area, high rigidity and suitable form of the particles, hydrophilic character and high permeability. The particle size and porosity are designed to maximize the surface area available for immobilization of ligand and consequently binding of target molecule. Supports in classical or low-performance affinity chromatography are usually non-rigid particles with large diameters like agarose, but also organic polymers like polyurethane or inorganic materials as large diameter silica particles (Hage, 2006). In column chromatography the adsorption rate is limited by slow particle diffusion for larger beads or low axial velocities and high pressure for smaller beads. The consequence is limited access for the biomolecules to small pores in the case of classical chromatographic supports. Good flow properties are desirable for rapid separations as they save considerable time during column equilibration, regeneration and cleaning (Champagne, 2007). The use of affinity supports with more rigid and effective materials that can be used in high performance liquid chromatography systems (HPLC) gives a technique known as highperformance affinity chromatography (Hage, 2006). In comparison with low pressure chromatography, HPLC is basically an improved form of the technique, where a mobile phase is forced through the column under high pressure instead of being allowed to drip through under gravity. Another improvement is an extremely sensitive detection system and complete automation of the process. Development of more resistant stationary phases has resulted in faster and better resolution and explains why HPLC became the most

powerful and versatile form of affinity chromatography (Wilson & Walker, 2005).

and to overcome the limitations of particulate stationary phase.

**<IP_ADDRESS> Natural supports**

Moreover, a chromatographic support should provide appropriate surface chemistry for the immobilization of affinity ligand and chemical stability for immobilization, adsorption, desorption and regeneration of the support itself (Champagne, 2007). Ability to withstand a wide range of thermal, mechanical, chemical and physical conditions and resistance to microbial and enzymatic cleavage are also desired (Tetala & van Beek, 2010). Different substances have been described and employed as affinity matrices to improve the separation

Natural polysaccharides, such as agarose and cellulose, possess highly ionic or carboxylate residues and they require processing before being suitable as affinity supports (Hermanson

retained target or the immobilized ligand (Tetala & van Beek, 2010).

**1.3.1 Supports in affinity chromatography**

### **1.3 Affinity chromatography**

Affinity chromatography is a purification technique that exploits the unique biological properties of biomolecules to bind ligands specifically and reversibly (Cuatrecasas, 1970). It is exceptional in purification science as a tool for highly selective isolation of antigens (drugs, hormones, peptides, proteins, viruses, cell components), antibodies, enzymes, sugars, glycoproteins and glycolipids, immunoglobulins, nucleotide-binding and metalbinding peptides and proteins (Mallik & Hage, 2006). The use of specific interactions makes affinity chromatography a powerful tool in isolation of a particular substance from a complex mixture. In theory, it is capable of giving absolute purification from a complex mixture in a single process and it eliminates the need for ammonium sulphate precipitation, ion exchange, and gel filtration steps in an isolation protocol (Hermanson et al., 1992).

Fig. 2. Principle of affinity chromatography: a) Equilibration of the stationary phase in a binding buffer. b) Sample is applied to the column. Target molecules will bind specifically but reversibly to the ligand, non-specifically bound contaminants will pass through in the flow through during washing with binding buffer. c) The purified compound is released from the biospecific ligand by elution using specific competition or non-specific change in buffer composition. d) Stationary phase is re-equilibrated with a binding buffer. The different stages of affinity chromatography are represented in a typical chromatogram in the right panel.

In affinity chromatography one of the two interacting molecules, called the affinity ligand, is immobilized on a stationary phase, either by covalent immobilization or by physical adsorption (Tetala & van Beek, 2010). The ligand can consist of an immobilized sequence of DNA or RNA, a protein or enzyme, lectin, amino acids, immunoglobulin, a biomimetic dye, an enzyme substrate or inhibitor, or a small molecule (Hage, 2006; Healthcare, 2007). Target molecules to be purified are applied in a mobile phase known as the application buffer to the column containing insoluble polymer or gel. The buffer is generally chosen to allow optimal binding of the immobilized ligand to its target while other sample components pass through with little or no retention (Mallik & Hage, 2006). Interaction between the ligand and the target molecule in a sample can be the result of electrostatic or hydrophobic interactions, van der Waals' forces or hydrogen bonding. The retained target molecule is then eluted and several different methods are possible. If a ligand has a weak or moderate affinity for the target molecules, the elution can be performed under isocratic conditions, where composition of the mobile phase remains unchanged during washing and elution. This technique is known as weak affinity chromatography. When a ligand has strong affinity to the target molecule, it can be released by changing the mobile phase or column conditions. For the most part, elution buffer differs from the application buffer in pH, ionic strength, temperature or polarity. This approach is known as nonspecific elution. Another possibility is biospecific elution, where a buffer with the same pH, ionic strength and polarity as the application buffer is used for elution but with a competing agent that can bind to either the retained target or the immobilized ligand (Tetala & van Beek, 2010).

### **1.3.1 Supports in affinity chromatography**

310 Affinity Chromatography

Affinity chromatography is a purification technique that exploits the unique biological properties of biomolecules to bind ligands specifically and reversibly (Cuatrecasas, 1970). It is exceptional in purification science as a tool for highly selective isolation of antigens (drugs, hormones, peptides, proteins, viruses, cell components), antibodies, enzymes, sugars, glycoproteins and glycolipids, immunoglobulins, nucleotide-binding and metalbinding peptides and proteins (Mallik & Hage, 2006). The use of specific interactions makes affinity chromatography a powerful tool in isolation of a particular substance from a complex mixture. In theory, it is capable of giving absolute purification from a complex mixture in a single process and it eliminates the need for ammonium sulphate precipitation, ion exchange, and gel filtration steps in an isolation protocol (Hermanson et al., 1992).

Fig. 2. Principle of affinity chromatography: a) Equilibration of the stationary phase in a binding buffer. b) Sample is applied to the column. Target molecules will bind specifically but reversibly to the ligand, non-specifically bound contaminants will pass through in the flow through during washing with binding buffer. c) The purified compound is released from the biospecific ligand by elution using specific competition or non-specific change in buffer composition. d) Stationary phase is re-equilibrated with a binding buffer. The different stages of affinity chromatography are represented in a typical chromatogram in the right panel.

In affinity chromatography one of the two interacting molecules, called the affinity ligand, is immobilized on a stationary phase, either by covalent immobilization or by physical adsorption (Tetala & van Beek, 2010). The ligand can consist of an immobilized sequence of DNA or RNA, a protein or enzyme, lectin, amino acids, immunoglobulin, a biomimetic dye, an enzyme substrate or inhibitor, or a small molecule (Hage, 2006; Healthcare, 2007). Target molecules to be purified are applied in a mobile phase known as the application buffer to the column containing insoluble polymer or gel. The buffer is generally chosen to allow optimal binding of the immobilized ligand to its target while other sample components pass through with little or no retention (Mallik & Hage, 2006). Interaction between the ligand and the target molecule in a sample can be the result of electrostatic or hydrophobic interactions, van der Waals' forces or hydrogen bonding. The retained target molecule is then eluted and several different methods are possible. If a ligand has a weak or moderate affinity for the target molecules, the elution can be performed under isocratic conditions, where

**1.3 Affinity chromatography**

The role of stationary phase or support in affinity chromatography is ligand immobilization (Hage, 2006). A support is any material to which a biospecific ligand may be covalently attached. Typically, the material to be used as an affinity support is insoluble in the system where the target molecule is found (Hermanson et al., 1992). The support main characteristics are: large specific area, high rigidity and suitable form of the particles, hydrophilic character and high permeability. The particle size and porosity are designed to maximize the surface area available for immobilization of ligand and consequently binding of target molecule. Supports in classical or low-performance affinity chromatography are usually non-rigid particles with large diameters like agarose, but also organic polymers like polyurethane or inorganic materials as large diameter silica particles (Hage, 2006). In column chromatography the adsorption rate is limited by slow particle diffusion for larger beads or low axial velocities and high pressure for smaller beads. The consequence is limited access for the biomolecules to small pores in the case of classical chromatographic supports. Good flow properties are desirable for rapid separations as they save considerable time during column equilibration, regeneration and cleaning (Champagne, 2007). The use of affinity supports with more rigid and effective materials that can be used in high performance liquid chromatography systems (HPLC) gives a technique known as highperformance affinity chromatography (Hage, 2006). In comparison with low pressure chromatography, HPLC is basically an improved form of the technique, where a mobile phase is forced through the column under high pressure instead of being allowed to drip through under gravity. Another improvement is an extremely sensitive detection system and complete automation of the process. Development of more resistant stationary phases has resulted in faster and better resolution and explains why HPLC became the most powerful and versatile form of affinity chromatography (Wilson & Walker, 2005).

Moreover, a chromatographic support should provide appropriate surface chemistry for the immobilization of affinity ligand and chemical stability for immobilization, adsorption, desorption and regeneration of the support itself (Champagne, 2007). Ability to withstand a wide range of thermal, mechanical, chemical and physical conditions and resistance to microbial and enzymatic cleavage are also desired (Tetala & van Beek, 2010). Different substances have been described and employed as affinity matrices to improve the separation and to overcome the limitations of particulate stationary phase.

#### **<IP_ADDRESS> Natural supports**

Natural polysaccharides, such as agarose and cellulose, possess highly ionic or carboxylate residues and they require processing before being suitable as affinity supports (Hermanson

The Value of Fungal Protease Inhibitors in Affinity Chromatography 313

Cyanogen Bromide (CNBr). At high pH CNBr introduces cyanate esters and imido carbonates into the matrix by reacting with the endogenous hydroxyl groups (Fig. 3). This activation works also with other synthetic polymers containing hydroxyl groups

Fig. 3. Mechanism of activation of polysaccharide matrix by CNBr and subsequent coupling

Alternative coupling procedures involve the use of bis-epoxides, N,N'-disubstituted carbodiimides, sulphonyl chloride, sodium periodate, N-hydroxysuccinimide esters and dichlorotriazines (Wilson & Walker, 2005). There are many different protocols for coupling a ligand to pre-activated supports and, as a consequence, it is easier to adjust the matrix and activation to suit the ligand than to adjust the ligand to suit the matrix and the activation.

The ligand is a molecule that binds reversibly to a specific molecule, enabling separation by affinity chromatography. When a suitable ligand is available for the protein of interest, high selectivity, resolution and capacity of affinity chromatography are expected. Successful affinity separation requires a biospecific ligand that can be covalently attached to a solid support and retain its specific binding affinity for the target molecule after unbound material is washed away. Furthermore, the bond between the ligand and the target molecule must be reversible to allow the target molecule to be eluted in an active form. The most commonly used biological ligands are antibodies, antigens, inhibitors, substrates, cofactors and coenzymes, lectins, protein A and protein G, and, among non-biological ligands, tyrazine dyes and metal-chelates (Hermanson et al., 1992; Mallik & Hage, 2006; Turkova,

The main criteria for the choice of an affinity ligand for target protein isolation are the functional groups of the ligand. To be immobilized, ligand must possess a functional group that will not be involved in the reversible binding of the ligand to the complementary target molecule, but which can be used to attach the ligand to the stationary support. The most common of such functional groups are -NH2, -COOH, -SH and –OH (Wilson & Walker, 2005). In addition, the use of a long spacer arm is indispensable in the case of low-molecular weight affinity ligands, to provide accessibility to the binding site of the target molecule. For effective chromatography, an equilibrium dissociation constant, *K*D, in the range of 10-4 to 10-

of amine-containing ligands (modified from Hermanson et al., 1992).

Moreover, many pre-activated matrices are commercially available.

**1.3.3 Ligand and ligand immobilization**

1993; Wilson & Walker, 2005).

(Hermanson et al., 1992).

et al., 1992). The most commonly used agarose is commercially available as a beaded form, known under the trademark Sepharose (GE Healthcare) and it has been further modified and developed to enhance properties required for an effective chromatographic material. It is an uncharged, hydrophilic matrix with an abundance of hydroxyl groups on the sugar residues which can be easily derivatized for covalent attachment of ligand (Healthcare, 2007). The primary structure of agarose consists of alternating residues of D-galactose and 3 anhydrogalactose. The secondary and tertiary structure forms a fabric with large accessible pores. The knitted porous structure of agarose is knotted at the juncture of the pores with strong hydrogen bonds. The large accessible pore structure of Sepharose yields affinity supports with sufficient capacity when the target molecule or ligand is a large protein or polysaccharide (Hermanson et al., 1992).

### **<IP_ADDRESS> Synthetic supports**

Synthetic supports, such as acrylamide derivatives, methacrylate derivatives, polystyrene and its derivatives and different membranes are produced by polymerization of functional monomers, to give matrices suitable for affinity-based separations. Commercially available synthetic materials have superior physical and chemical durability and can withstand the process of separation better than the natural soft gels. Synthetic supports are made with monomers that contain primary or secondary hydroxyl groups that maintain hydrophilicity and allow compatibility with most coupling methods (Hermanson et al., 1992).

### **<IP_ADDRESS> Monolithic supports**

To overcome the difficulties of slow mass transfer in different synthetic supports, a new generation of monolithic solid supports was introduced in affinity chromatography. Monolith is a name for the chromatographic stationary phase consisting of a single piece of highly porous material (Barut et al., 2008). They are prepared in various dimensions with agglomeration-type or fibrous microstructures. In recent years, the polymeric macroporous material, based on radical co-polymerization of glycidyl methacrylate and ethylene glycol dimethacrylate (GMA/EDMA) has been introduced under the trademark CIM® (Convective Interaction Media). There are several reasons for the popularity of monolithic supports in HPLC and affinity chromatography. The main advantage of CIM monolithic systems versus traditional chromatographic supports are much better mass transfer properties, ease of use, the ability to be manufactured with a wide range of pore sizes and shapes, simple scaling up and scaling down, and low back pressure even at very high volumetric flow rates (up to 10 column volumes/min) without the loss of efficiency and capacity (Podgornik & Štrancar, 2005). Monolithic supports can carry many specific ligands for affinity chromatography and the vast potential of monoliths for immobilization of affinity ligands has been recognized, resulting in an increase in experience in applying this technology to bioanalytical and biotechnological activities (Brne et al., 2009; Champagne, 2007; Švec et al., 2003).

### **1.3.2 Support activation**

Support activation is the first step of ligand immobilization to a chromatographic support. It is a process of chemically modifying the support in a way to form a covalent bond with the ligand of choice. The reaction conditions and the proportion of the reagents will determine the number of ligand molecules that can be attached to the support surface (Wilson & Walker, 2005). The most common activation method for polysaccharide supports is with

et al., 1992). The most commonly used agarose is commercially available as a beaded form, known under the trademark Sepharose (GE Healthcare) and it has been further modified and developed to enhance properties required for an effective chromatographic material. It is an uncharged, hydrophilic matrix with an abundance of hydroxyl groups on the sugar residues which can be easily derivatized for covalent attachment of ligand (Healthcare, 2007). The primary structure of agarose consists of alternating residues of D-galactose and 3 anhydrogalactose. The secondary and tertiary structure forms a fabric with large accessible pores. The knitted porous structure of agarose is knotted at the juncture of the pores with strong hydrogen bonds. The large accessible pore structure of Sepharose yields affinity supports with sufficient capacity when the target molecule or ligand is a large protein or

Synthetic supports, such as acrylamide derivatives, methacrylate derivatives, polystyrene and its derivatives and different membranes are produced by polymerization of functional monomers, to give matrices suitable for affinity-based separations. Commercially available synthetic materials have superior physical and chemical durability and can withstand the process of separation better than the natural soft gels. Synthetic supports are made with monomers that contain primary or secondary hydroxyl groups that maintain hydrophilicity

To overcome the difficulties of slow mass transfer in different synthetic supports, a new generation of monolithic solid supports was introduced in affinity chromatography. Monolith is a name for the chromatographic stationary phase consisting of a single piece of highly porous material (Barut et al., 2008). They are prepared in various dimensions with agglomeration-type or fibrous microstructures. In recent years, the polymeric macroporous material, based on radical co-polymerization of glycidyl methacrylate and ethylene glycol dimethacrylate (GMA/EDMA) has been introduced under the trademark CIM® (Convective Interaction Media). There are several reasons for the popularity of monolithic supports in HPLC and affinity chromatography. The main advantage of CIM monolithic systems versus traditional chromatographic supports are much better mass transfer properties, ease of use, the ability to be manufactured with a wide range of pore sizes and shapes, simple scaling up and scaling down, and low back pressure even at very high volumetric flow rates (up to 10 column volumes/min) without the loss of efficiency and capacity (Podgornik & Štrancar, 2005). Monolithic supports can carry many specific ligands for affinity chromatography and the vast potential of monoliths for immobilization of affinity ligands has been recognized, resulting in an increase in experience in applying this technology to bioanalytical and

and allow compatibility with most coupling methods (Hermanson et al., 1992).

biotechnological activities (Brne et al., 2009; Champagne, 2007; Švec et al., 2003).

Support activation is the first step of ligand immobilization to a chromatographic support. It is a process of chemically modifying the support in a way to form a covalent bond with the ligand of choice. The reaction conditions and the proportion of the reagents will determine the number of ligand molecules that can be attached to the support surface (Wilson & Walker, 2005). The most common activation method for polysaccharide supports is with

polysaccharide (Hermanson et al., 1992).

**<IP_ADDRESS> Synthetic supports**

**<IP_ADDRESS> Monolithic supports**

**1.3.2 Support activation**

Cyanogen Bromide (CNBr). At high pH CNBr introduces cyanate esters and imido carbonates into the matrix by reacting with the endogenous hydroxyl groups (Fig. 3). This activation works also with other synthetic polymers containing hydroxyl groups (Hermanson et al., 1992).

Fig. 3. Mechanism of activation of polysaccharide matrix by CNBr and subsequent coupling of amine-containing ligands (modified from Hermanson et al., 1992).

Alternative coupling procedures involve the use of bis-epoxides, N,N'-disubstituted carbodiimides, sulphonyl chloride, sodium periodate, N-hydroxysuccinimide esters and dichlorotriazines (Wilson & Walker, 2005). There are many different protocols for coupling a ligand to pre-activated supports and, as a consequence, it is easier to adjust the matrix and activation to suit the ligand than to adjust the ligand to suit the matrix and the activation. Moreover, many pre-activated matrices are commercially available.

### **1.3.3 Ligand and ligand immobilization**

The ligand is a molecule that binds reversibly to a specific molecule, enabling separation by affinity chromatography. When a suitable ligand is available for the protein of interest, high selectivity, resolution and capacity of affinity chromatography are expected. Successful affinity separation requires a biospecific ligand that can be covalently attached to a solid support and retain its specific binding affinity for the target molecule after unbound material is washed away. Furthermore, the bond between the ligand and the target molecule must be reversible to allow the target molecule to be eluted in an active form. The most commonly used biological ligands are antibodies, antigens, inhibitors, substrates, cofactors and coenzymes, lectins, protein A and protein G, and, among non-biological ligands, tyrazine dyes and metal-chelates (Hermanson et al., 1992; Mallik & Hage, 2006; Turkova, 1993; Wilson & Walker, 2005).

The main criteria for the choice of an affinity ligand for target protein isolation are the functional groups of the ligand. To be immobilized, ligand must possess a functional group that will not be involved in the reversible binding of the ligand to the complementary target molecule, but which can be used to attach the ligand to the stationary support. The most common of such functional groups are -NH2, -COOH, -SH and –OH (Wilson & Walker, 2005). In addition, the use of a long spacer arm is indispensable in the case of low-molecular weight affinity ligands, to provide accessibility to the binding site of the target molecule. For effective chromatography, an equilibrium dissociation constant, *K*D, in the range of 10-4 to 10-

The Value of Fungal Protease Inhibitors in Affinity Chromatography 315

themselves, but in practice, substrates are immediately converted to product after contact with the target enzyme (Healthcare, 2007; Polanowski et al., 2003). Therefore, the best results

Synthetic inhibitors present effective ligands for many proteases. The synthetic inhibitor para-aminobenzamidine is used as affinity ligand for trypsin, trypsin-like serine proteases and zymogens. Benzamidine-Sepharose is commercially available and frequently used for removing molecules from cell culture supernatants, bacterial lysates or serum (Healthcare, 2007). Bovine basic pancreatic trypsin inhibitor (BPTI), or aprotinin, and inhibitors from legume seeds, such as soybean trypsin inhibitor (SBTI), are frequently employed protein ligands for serine protease isolation (Hewlett, 1990; Polanowski et al., 2003). Group specific inhibitors, that are selective and able to react with a protease from single class, are also effective ligands for specific classes of proteases. Egg white cystatin C coupled to Sepharose allows selective isolation of cysteine proteases from tissue or cell extracts, biological fluids and culture media (Tombaccini et al., 2001). Pepstatin A has been employed as a universal means of purifying aspartic proteases from a variety of sources. An example is isolation of an aspartic protease family from wild growing basidiomycete *Clitocybe nebularis* (Sabotič et

The protease inhibitors from mushrooms, cnispin and macrocypin, were selected as examples of application of fungal protease inhibitors in affinity chromatography for

Recombinant protein protease inhibitors cnispin from *Clitocybe nebularis* and macrocypin from *Macrolepiota procera* were heterologously expressed in *Escherichia coli* BL21(DE3), refolded from inclusion bodies and purified as previously described (Avanzo et al., 2009;

Recombinant cnispin and macrocypin were immobilized to the CNBr-activated Sepharose (GE Healthcare) according to the manufacturer's recommendations. For each ligand a 15 ml Sepharose column was prepared. The inhibitory activity of the Sepharose immobilized protease inhibitors was confirmed by a trypsin inhibition assay for cnispin and papain inhibition assay for macrocypin as described (Avanzo et al., 2009; Sabotič et al., 2009b).

Convective Interaction Media (CIM) epoxy and CIM carbonyldiimidazole (CDI) disks with a diameter of 12 mm and thickness of 3 mm, equipped with a special cartridge, were obtained from BIA Separations (Ljubljana, Slovenia). The CIM epoxy monoliths are synthesized from glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) monomers in the presence of pore forming solvents dodecanol and cyclohexanol. The resulting rigid monolith bears epoxy groups. CDI monoliths are prepared from epoxy monoliths hydrolyzed to obtain

isolation of proteases. Similar protocols can be followed for other protease inhibitors.

in protease purification may be gained by using highly specific inhibitors.

al., 2009a).

**2. Materials and methods**

**2.1 Preparation of ligands**

**2.2 Immobilization of ligands to the Sepharose matrix**

**2.3 Immobilization of ligands to monolith supports**

Sabotič et al., 2009b).

8 M in free solution is required for successful separation (Healthcare, 2007; Turkova, 1993). When the dissociation constant is outside this range, altering elution methods may however help to execute successful affinity chromatography (Healthcare, 2007).

Ligands in biospecific chromatography can be classified into two types, monospecific and group specific. Monospecific ligands are those that bind only one molecule; an example is monoclonal antibody to a protein. In this case, a monospecific ligand is expected to bind the target molecules with large association constant, while binding weakly or not at all, to others (Miller, 2005). Group specific ligands have an affinity for a group of related substances rather than for a single type of molecule. The specificity derives from the selectivity of the ligand and the use of selective elution conditions. For example, a lectin can be used for affinity purification of glycoproteins, polysaccharides and glycolipids with the same glyco-signature (Healthcare, 2007; Miller, 2005). The plant lectin isolated from jack bean (*Canavalia ensiformis*) Concanavalin A (ConA) is a routinely used group specific ligand (Healthcare, 2007).

Several approaches have been reported for placing ligands within supports for chromatography. Examples include various covalent immobilization methods, as well as biospecific adsorption, entrapment and cross-linking. Covalent binding is preferred among the other immobilization methods, because it prevents leaking and combines the high selectivity of the reaction with the chemical and mechanical properties of the support (Benčina et al., 2004).

Introducing a spacer arm minimizes the risk of steric interference such as binding between support and target molecule or low accessibility of the ligand. Spacer arms are lowmolecular-weight molecules interposed between the ligand and the solid support. They usually consist of linear hydrocarbon chains with functionalities on both ends for easy coupling to the support and ligand. One end of the spacer is immobilized to the matrix using traditional immobilization chemistries, while the other end is connected to the ligand, using a secondary coupling procedure. The result is immobilized ligand that sticks out from the matrix by a distance equal to the length of spacer arm chosen, where the optimal length is up to 10 carbon atoms (Hermanson et al., 1992). In addition, ligands involved in interaction must be sufficiently distant from the solid support to minimize steric interference with the binding processes (Cuatrecasas et al., 1968; Hermanson et al., 1992). With rigid support materials, a spacer molecule may also provide greater flexibility, allowing the immobilized ligand to move into position to establish the correct binding orientation with a protein. Sometimes the chemical structure of a spacer arm is critical for the success of separation. It is also important to consider the hydrophilicity of the spacer molecule, as some spacers are purely hydrophobic, for example methylene groups; others are hydrophilic, possessing carbonyl or imido groups (Wilson & Walker, 2005). Several ready to use supports of agarose, cellulose and polyacrylamide with a variety of spacer arms and pre-attached ligands are commercially available.

### **1.4 Affinity chromatography for purification of proteases**

For the affinity chromatography of proteases different ligands from substrates or their analogues to various synthetic or naturally occurring inhibitors have been immobilized. Appropriate ligands for protease purification can be immobilized enzyme substrates themselves, but in practice, substrates are immediately converted to product after contact with the target enzyme (Healthcare, 2007; Polanowski et al., 2003). Therefore, the best results in protease purification may be gained by using highly specific inhibitors.

Synthetic inhibitors present effective ligands for many proteases. The synthetic inhibitor para-aminobenzamidine is used as affinity ligand for trypsin, trypsin-like serine proteases and zymogens. Benzamidine-Sepharose is commercially available and frequently used for removing molecules from cell culture supernatants, bacterial lysates or serum (Healthcare, 2007). Bovine basic pancreatic trypsin inhibitor (BPTI), or aprotinin, and inhibitors from legume seeds, such as soybean trypsin inhibitor (SBTI), are frequently employed protein ligands for serine protease isolation (Hewlett, 1990; Polanowski et al., 2003). Group specific inhibitors, that are selective and able to react with a protease from single class, are also effective ligands for specific classes of proteases. Egg white cystatin C coupled to Sepharose allows selective isolation of cysteine proteases from tissue or cell extracts, biological fluids and culture media (Tombaccini et al., 2001). Pepstatin A has been employed as a universal means of purifying aspartic proteases from a variety of sources. An example is isolation of an aspartic protease family from wild growing basidiomycete *Clitocybe nebularis* (Sabotič et al., 2009a).

### **2. Materials and methods**

314 Affinity Chromatography

8 M in free solution is required for successful separation (Healthcare, 2007; Turkova, 1993). When the dissociation constant is outside this range, altering elution methods may however

Ligands in biospecific chromatography can be classified into two types, monospecific and group specific. Monospecific ligands are those that bind only one molecule; an example is monoclonal antibody to a protein. In this case, a monospecific ligand is expected to bind the target molecules with large association constant, while binding weakly or not at all, to others (Miller, 2005). Group specific ligands have an affinity for a group of related substances rather than for a single type of molecule. The specificity derives from the selectivity of the ligand and the use of selective elution conditions. For example, a lectin can be used for affinity purification of glycoproteins, polysaccharides and glycolipids with the same glyco-signature (Healthcare, 2007; Miller, 2005). The plant lectin isolated from jack bean (*Canavalia ensiformis*) Concanavalin A (ConA) is a routinely used group specific ligand

Several approaches have been reported for placing ligands within supports for chromatography. Examples include various covalent immobilization methods, as well as biospecific adsorption, entrapment and cross-linking. Covalent binding is preferred among the other immobilization methods, because it prevents leaking and combines the high selectivity of the reaction with the chemical and mechanical properties of the support

Introducing a spacer arm minimizes the risk of steric interference such as binding between support and target molecule or low accessibility of the ligand. Spacer arms are lowmolecular-weight molecules interposed between the ligand and the solid support. They usually consist of linear hydrocarbon chains with functionalities on both ends for easy coupling to the support and ligand. One end of the spacer is immobilized to the matrix using traditional immobilization chemistries, while the other end is connected to the ligand, using a secondary coupling procedure. The result is immobilized ligand that sticks out from the matrix by a distance equal to the length of spacer arm chosen, where the optimal length is up to 10 carbon atoms (Hermanson et al., 1992). In addition, ligands involved in interaction must be sufficiently distant from the solid support to minimize steric interference with the binding processes (Cuatrecasas et al., 1968; Hermanson et al., 1992). With rigid support materials, a spacer molecule may also provide greater flexibility, allowing the immobilized ligand to move into position to establish the correct binding orientation with a protein. Sometimes the chemical structure of a spacer arm is critical for the success of separation. It is also important to consider the hydrophilicity of the spacer molecule, as some spacers are purely hydrophobic, for example methylene groups; others are hydrophilic, possessing carbonyl or imido groups (Wilson & Walker, 2005). Several ready to use supports of agarose, cellulose and polyacrylamide with a variety of spacer arms and

For the affinity chromatography of proteases different ligands from substrates or their analogues to various synthetic or naturally occurring inhibitors have been immobilized. Appropriate ligands for protease purification can be immobilized enzyme substrates

help to execute successful affinity chromatography (Healthcare, 2007).

(Healthcare, 2007).

(Benčina et al., 2004).

pre-attached ligands are commercially available.

**1.4 Affinity chromatography for purification of proteases**

The protease inhibitors from mushrooms, cnispin and macrocypin, were selected as examples of application of fungal protease inhibitors in affinity chromatography for isolation of proteases. Similar protocols can be followed for other protease inhibitors.

### **2.1 Preparation of ligands**

Recombinant protein protease inhibitors cnispin from *Clitocybe nebularis* and macrocypin from *Macrolepiota procera* were heterologously expressed in *Escherichia coli* BL21(DE3), refolded from inclusion bodies and purified as previously described (Avanzo et al., 2009; Sabotič et al., 2009b).

### **2.2 Immobilization of ligands to the Sepharose matrix**

Recombinant cnispin and macrocypin were immobilized to the CNBr-activated Sepharose (GE Healthcare) according to the manufacturer's recommendations. For each ligand a 15 ml Sepharose column was prepared. The inhibitory activity of the Sepharose immobilized protease inhibitors was confirmed by a trypsin inhibition assay for cnispin and papain inhibition assay for macrocypin as described (Avanzo et al., 2009; Sabotič et al., 2009b).

### **2.3 Immobilization of ligands to monolith supports**

Convective Interaction Media (CIM) epoxy and CIM carbonyldiimidazole (CDI) disks with a diameter of 12 mm and thickness of 3 mm, equipped with a special cartridge, were obtained from BIA Separations (Ljubljana, Slovenia). The CIM epoxy monoliths are synthesized from glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) monomers in the presence of pore forming solvents dodecanol and cyclohexanol. The resulting rigid monolith bears epoxy groups. CDI monoliths are prepared from epoxy monoliths hydrolyzed to obtain

The Value of Fungal Protease Inhibitors in Affinity Chromatography 317

Kiwi fruits (*Actinidia deliciosa*), previously stored at -20°C, were thawed and homogenized in 0.1 M Na acetate buffer pH 6.5 with 0.1 % Na2S2O3 and 0.3 M NaCl. Before application to the affinity column the crude protein extract was cleared by centrifugation for 15 min at 16000 g

Bean seeds (*Phaseolus vulgaris* L.) were germinated aseptically at 28°C in the dark. After 3 days the cuticle was removed and the seeds homogenized at 4°C in 0.1 M Na acetate buffer, pH 6.5 supplemented with 0.5 M NaCl, 1.5 mM EDTA and 2 mM DTT using an ultraturax homogenizer (IKA-Labortechnik). The insoluble material was removed by centrifugation for 30 min at 12000 g and 4°C. Proteins in the supernatant were precipitated by ammonium sulphate at 80% saturation overnight at 4°C. The precipitate was collected by centrifugation (20 min, 16000 g), dissolved in 0.1 M Na acetate buffer, 0.5 M NaCl, pH 6.5, and dialyzed (7 kDa cut-off) against the same buffer. The extract was cleared by centrifugation (30 min, 16000 g) and applied to a Sephacryl S200 column (GE Healthcare) equilibrated with 0.1 M Na acetate, 0.5 M NaCl, pH 6.5 for size exclusion chromatography. Protein containing fractions (determined by measurement of absorbance at 280 nm) were pooled and

Cortical tissue was dissected from pig kidney and homogenized in 0.1 M phosphate buffer, pH 6 supplemented with 0.3 M NaCl, 60 mM EDTA and 15 mM DTT using an ultraturax homogenizer (IKA-Labortechnik). The insoluble material was removed by centrifugation at 4 °C for 20 min at 8000 g and the supernatant cleared by centrifugation for 20 min at 16000 g

*Bacillus subtilis* was cultivated (0.5 % yeast extract, 1 % powdered milk) at 37°C and 220 rpm for 24 h. Bacteria were removed by centrifugation at 7000 g for 20 min at 4°C. Proteins in the culture supernatant were precipitated with ammonium sulphate at 80% saturation overnight at 4°C. After dialysis against 0.1 M Na acetate, 0.3 M NaCl, 1 mM EDTA, pH 6.5,

Partially purified bovine trypsin and chymotrypsin mixture (Fluka) was dissolved at 1 mg/ml in 0.1 M Na acetate, 0.3 M NaCl, 1 mM EDTA, pH 6.5. Papain from *Carica papaya*

A cnispin-affinity column was used to isolate subtilisin from the *B. subtilis* culture supernatant and trypsin from the partially purified bovine serine protease mixture.

the protein extract was cleared by centrifugation at 16000 g for 10 min at 4°C.

(Sigma) was dissolved at 1 mg/ml in 0.1 M Tris-HCl, 0.6 M NaCl, pH 7.0.

**2.4.1 Kiwi fruit**

**2.4.2 Germinated bean seeds**

**2.4.3 Pig kidney cortex**

and 4°C.

**2.4.5 Enzymes**

**2.5 Affinity chromatography**

**2.5.1 Sepharose matrix affinity chromatography**

concentrated by ultrafiltration (UM-10, Amicon).

**2.4.4** *Bacillus subtilis* **culture supernatant**

and 4°C.

hydroxyl group, which are then treated with 1,1'carbonyldiimidazole (Benčina et al., 2004). Macrocypin was immobilized on CIM monoliths using these two different activation chemistries and, additionally, to a monolith that contained short functional spacers (see 2.3.3).

### **2.3.1 Immobilization on a CIM epoxy disk**

Macrocypin was covalently immobilized to the original epoxy groups of the GMA/EDMA material. After conditioning the CIM epoxy disk with 0.5 M phosphate buffer pH 7.0 for 1 h, 2 ml of 2.5 mg/ml macrocypin in the same buffer was applied to the disk with a syringe. The monolithic disk was then incubated for 24 h at pH 7.0 at 45°C with gentle shaking. Afterwards, the monolith was washed for 1 h with water to remove unbound material, and then incubated for 1 h in 0.5 M H2SO4 at 50°C to inactivate the remaining epoxy groups. The disk was then washed with the mobile phase 0.1 M Tris-HCl, 0.6 M NaCl, pH 7.0 for 1 h at 1 ml/min flow, and stored in the same buffer at 4°C.

### **2.3.2 Immobilization on a CIM CDI disk**

Macrocypin was covalently immobilized to a CIM CDI disk. The disk was first washed with 0.5 M phosphate buffer pH 7 for 1 h, and then permeated with 2 ml of macrocypin at 2.5 mg/ml in the same phosphate buffer. Afterwards, the disk was incubated with macrocypin solution for 24 h at pH 7.0 at 45°C under gentle shaking. The disk was then rinsed with distilled water for 1 h at 1 ml/min flow and stored in 0.5 M phosphate buffer pH 7 at 4°C.

### **2.3.3 Immobilization on CIM disk by a short spacer arm**

A CIM ethylene diamine (EDA) disk containing (free) amine groups was first incubated in 50 mM phosphate buffer, pH 8.0 for 1 h and then derivatized with 10% (v/v) glutaraldehyde solution in the same buffer overnight in the dark at room temperature and under gentle shaking. The disk was washed with 50 mM phosphate buffer, pH 8.0 to remove the reagent and then with 0.5 mM phosphate buffer, pH 3.0. To immobilize macrocypin, the derivatized disk was permeated with 1 ml of macrocypin (2.5 mg/ml in 50 mM phosphate buffer pH 3.0) and allowed to react for 24 h in the dark, at room temperature and under gentle shaking. The disk was then washed with 0.5 M phosphate buffer, pH 3.0 for 1h. To reduce the formed Schiff's bases, the disk was additionally washed with 0.1 M sodium cyanoborohydride in 0.5 M phosphate buffer, pH 8.0, for 2 h. Free remaining groups were end-capped with 1 M monoethanolamine in phosphate buffer (0.5 M, pH 8.0) for 3 h. Finally, the disk was washed with distilled water for 1 h at 1 ml/min flow and stored in 0.01 M phosphate buffer at 4°C.

### **2.4 Protein extracts and enzymes**

Crude protein extracts of papain-like cysteine proteases and legumain were prepared from plant (kiwi fruit and germinated bean seeds) and animal (pig kidney cortex) sources, and of the appropriate bacterial source for isolation of serine proteases. The above natural sources were selected based on previous experience and literature data as appropriate sources for target proteases. In addition, partially purified, commercially available bovine serine proteases and purified plant papain were used for characterization of the prepared affinity columns. The extracts were prepared in the appropriate binding buffers for direct application to the affinity chromatography column.

### **2.4.1 Kiwi fruit**

316 Affinity Chromatography

hydroxyl group, which are then treated with 1,1'carbonyldiimidazole (Benčina et al., 2004). Macrocypin was immobilized on CIM monoliths using these two different activation chemistries and, additionally, to a monolith that contained short functional spacers (see 2.3.3).

Macrocypin was covalently immobilized to the original epoxy groups of the GMA/EDMA material. After conditioning the CIM epoxy disk with 0.5 M phosphate buffer pH 7.0 for 1 h, 2 ml of 2.5 mg/ml macrocypin in the same buffer was applied to the disk with a syringe. The monolithic disk was then incubated for 24 h at pH 7.0 at 45°C with gentle shaking. Afterwards, the monolith was washed for 1 h with water to remove unbound material, and then incubated for 1 h in 0.5 M H2SO4 at 50°C to inactivate the remaining epoxy groups. The disk was then washed with the mobile phase 0.1 M Tris-HCl, 0.6 M NaCl, pH 7.0 for 1 h at 1

Macrocypin was covalently immobilized to a CIM CDI disk. The disk was first washed with 0.5 M phosphate buffer pH 7 for 1 h, and then permeated with 2 ml of macrocypin at 2.5 mg/ml in the same phosphate buffer. Afterwards, the disk was incubated with macrocypin solution for 24 h at pH 7.0 at 45°C under gentle shaking. The disk was then rinsed with distilled water for 1 h at 1 ml/min flow and stored in 0.5 M phosphate buffer pH 7 at 4°C.

A CIM ethylene diamine (EDA) disk containing (free) amine groups was first incubated in 50 mM phosphate buffer, pH 8.0 for 1 h and then derivatized with 10% (v/v) glutaraldehyde solution in the same buffer overnight in the dark at room temperature and under gentle shaking. The disk was washed with 50 mM phosphate buffer, pH 8.0 to remove the reagent and then with 0.5 mM phosphate buffer, pH 3.0. To immobilize macrocypin, the derivatized disk was permeated with 1 ml of macrocypin (2.5 mg/ml in 50 mM phosphate buffer pH 3.0) and allowed to react for 24 h in the dark, at room temperature and under gentle shaking. The disk was then washed with 0.5 M phosphate buffer, pH 3.0 for 1h. To reduce the formed Schiff's bases, the disk was additionally washed with 0.1 M sodium cyanoborohydride in 0.5 M phosphate buffer, pH 8.0, for 2 h. Free remaining groups were end-capped with 1 M monoethanolamine in phosphate buffer (0.5 M, pH 8.0) for 3 h. Finally, the disk was washed with distilled water for 1 h at 1 ml/min flow and stored in 0.01 M phosphate buffer at 4°C.

Crude protein extracts of papain-like cysteine proteases and legumain were prepared from plant (kiwi fruit and germinated bean seeds) and animal (pig kidney cortex) sources, and of the appropriate bacterial source for isolation of serine proteases. The above natural sources were selected based on previous experience and literature data as appropriate sources for target proteases. In addition, partially purified, commercially available bovine serine proteases and purified plant papain were used for characterization of the prepared affinity columns. The extracts were prepared in the appropriate binding buffers for direct

**2.3.1 Immobilization on a CIM epoxy disk**

ml/min flow, and stored in the same buffer at 4°C.

**2.3.3 Immobilization on CIM disk by a short spacer arm**

**2.3.2 Immobilization on a CIM CDI disk**

**2.4 Protein extracts and enzymes**

application to the affinity chromatography column.

Kiwi fruits (*Actinidia deliciosa*), previously stored at -20°C, were thawed and homogenized in 0.1 M Na acetate buffer pH 6.5 with 0.1 % Na2S2O3 and 0.3 M NaCl. Before application to the affinity column the crude protein extract was cleared by centrifugation for 15 min at 16000 g and 4°C.

### **2.4.2 Germinated bean seeds**

Bean seeds (*Phaseolus vulgaris* L.) were germinated aseptically at 28°C in the dark. After 3 days the cuticle was removed and the seeds homogenized at 4°C in 0.1 M Na acetate buffer, pH 6.5 supplemented with 0.5 M NaCl, 1.5 mM EDTA and 2 mM DTT using an ultraturax homogenizer (IKA-Labortechnik). The insoluble material was removed by centrifugation for 30 min at 12000 g and 4°C. Proteins in the supernatant were precipitated by ammonium sulphate at 80% saturation overnight at 4°C. The precipitate was collected by centrifugation (20 min, 16000 g), dissolved in 0.1 M Na acetate buffer, 0.5 M NaCl, pH 6.5, and dialyzed (7 kDa cut-off) against the same buffer. The extract was cleared by centrifugation (30 min, 16000 g) and applied to a Sephacryl S200 column (GE Healthcare) equilibrated with 0.1 M Na acetate, 0.5 M NaCl, pH 6.5 for size exclusion chromatography. Protein containing fractions (determined by measurement of absorbance at 280 nm) were pooled and concentrated by ultrafiltration (UM-10, Amicon).

### **2.4.3 Pig kidney cortex**