**3.4 Proteases**

The zymography may also be applied for proteases. It is a simple, quantitative and functional technique to analyze the activity of proteases (Leber & Balkwill, 1997). It consists basically of two stages, the separation through electrophoresis, followed by the activity detection of the enzyme in polyacrylamide gel, in non-reducing conditions (without treatment with DTT or β-mercaptoethanol) (Dong-Min et al., 2011). This technique has been used to evaluate the level of proteases in tissues or biological fluids, and it bears the advantage of distinguishing different kinds of enzymes due to the characteristic of mobility that each enzyme presents (Raser et al., 1995). The protease activity in zymography is observed as a clear band, indicating the substrate proteolysis after colored with Coomassie Brilliant Blue (Kim et al., 1998).

This methodology is widely employed for the detection of Matrix metalloproteinases (MMPs). However, it can also be employed for other types of proteases, with the need of adjustments in the methodology, such as the substitution of the substrate, generally gelatin for casein. Unfortunately, the zymography with casein is very little sensitive, when compared to the zymography with gelatin. Besides, casein migrates in the gel during the electrophoresis due to its relative low molar mass. That results in two clearly defined areas in the gel: the upper part, which still contains excess casein and the lower part, with less casein (Beurden and Von denHoff, 2005).

For the detection of proteases (Fig. 2E), the sample must be diluted in the sample buffer (5x) of the gel (0.4M Tris-HCl, pH 6.8; 5% SDS; 20% glycerol 0.03% Bromophenol blue). The samples cannot be boiled, because this process denatures the enzyme and it will no longer present activity (Kleiner & Stetler-Stevenson, 1994). The electrophoresis of the samples containing the protease must be performed according to Laemmli (1970). The gel concentration must be prepared according to the molar mass of its protease. The electrophoresis may be performed in constant 100V for 1-2 hours at 4°C.

For the development of the proteolytic activity, the gel must be incubated with 70 mL of buffer with the appropriate reaction pH, for 5 min., 4°C, 100 rpm. Following, the buffer must be removed and the gel must be incubated with 70 mL of Triton X-100 2.5% prepared in the reaction buffer. The gel must be kept at 100 rpm, for 30 min, 4°C. This step is for the removal of the SDS and the activation of the protease. Afterwards, the excess Triton X-100 must be

and without boiling. The advantage of the activity detection through zymograms is that effectively all active isoforms present there may be detected, once that regardless on the

In this case, the SDS gel must be performed with the running buffer commonly adopted in the protocols and, after the electrophoresis run, the proteins will have to be transferred to another gel composed by agarose and xylan, a substrate that is specific to the activity to be detected. Such transferring happens when there is a kind of "sandwich" with the polyacrylamide gel and the agarose + substrate gel. The transferring must happen overnight. After this period, the agarose + substrate gel, now also with the proteins to be analyzed, will have to be incubated in the buffer that is suitable for the isoforms under study, during one hour, following with the coloration suitable for the activity detection of

The zymography may also be applied for proteases. It is a simple, quantitative and functional technique to analyze the activity of proteases (Leber & Balkwill, 1997). It consists basically of two stages, the separation through electrophoresis, followed by the activity detection of the enzyme in polyacrylamide gel, in non-reducing conditions (without treatment with DTT or β-mercaptoethanol) (Dong-Min et al., 2011). This technique has been used to evaluate the level of proteases in tissues or biological fluids, and it bears the advantage of distinguishing different kinds of enzymes due to the characteristic of mobility that each enzyme presents (Raser et al., 1995). The protease activity in zymography is observed as a clear band, indicating the substrate proteolysis after colored with Coomassie

This methodology is widely employed for the detection of Matrix metalloproteinases (MMPs). However, it can also be employed for other types of proteases, with the need of adjustments in the methodology, such as the substitution of the substrate, generally gelatin for casein. Unfortunately, the zymography with casein is very little sensitive, when compared to the zymography with gelatin. Besides, casein migrates in the gel during the electrophoresis due to its relative low molar mass. That results in two clearly defined areas in the gel: the upper part, which still contains excess casein and the lower part, with less

For the detection of proteases (Fig. 2E), the sample must be diluted in the sample buffer (5x) of the gel (0.4M Tris-HCl, pH 6.8; 5% SDS; 20% glycerol 0.03% Bromophenol blue). The samples cannot be boiled, because this process denatures the enzyme and it will no longer present activity (Kleiner & Stetler-Stevenson, 1994). The electrophoresis of the samples containing the protease must be performed according to Laemmli (1970). The gel concentration must be prepared according to the molar mass of its protease. The

For the development of the proteolytic activity, the gel must be incubated with 70 mL of buffer with the appropriate reaction pH, for 5 min., 4°C, 100 rpm. Following, the buffer must be removed and the gel must be incubated with 70 mL of Triton X-100 2.5% prepared in the reaction buffer. The gel must be kept at 100 rpm, for 30 min, 4°C. This step is for the removal of the SDS and the activation of the protease. Afterwards, the excess Triton X-100 must be

electrophoresis may be performed in constant 100V for 1-2 hours at 4°C.

isoelectric point, all proteins will migrate in the gel, because of their molar mass.

the enzyme analyzed.

Brilliant Blue (Kim et al., 1998).

casein (Beurden and Von denHoff, 2005).

**3.4 Proteases**

removed. For so, add 70 mL of buffer with the appropriate reaction pH, incubate for 30 min., at 4°C, 100rpm.

Fig. 2. Activity gels for different enzymes in SDS-PAGE 10%. (A1) α-glucosidase (a type of amylase) revealed with Coomassie Blue; (A2) α-glucosidase revealed with 10 mM Iodine solution and 14 mM potassium iodide; (B1) polygalacturonase revealed with silver solution; (B2) polygalacturonase activity revealed with 0.02% ruthenium red; (C1) xylanase revealed with silver solution; (C2) xylanase activity revealed with Congo red; (D1) Zymogram for xylanase revealed with silver solution and (D2) Congo red; (E) protease activity revealed with Coomassie Blue.

Following, remove the buffer and add the casein solution at 3%, prepared in a buffer with the enzyme reaction pH. The gel must be incubated for 30 min, at 4ºC, for the diffusion of the casein to the gel. After that, the gel must be bathed at the enzyme reaction temperature for the period of 1-2 hours, so that the enzymatic reaction occurs (such period may be adjusted according to each enzyme and concentration).

The excess casein must be removed by bathing the gel for 5 times with distilled water at room temperature (García-Carreño et al., 1993 and Kleiner & Stetler-Steveson, 1994) with modifications.

For the coloration, the gel must be stained with a solution containing 40% ethanol, 10% acetic acid and 0.1% Coomassie brilliant blue R-250 (García-Carreño et al., 1993). In this stage, the gel shows a blue bottom and where the hydrolysis took place, there will be a white halo. The gel needs to have the excess Coomassie brilliant blue removed; hence, it will have to be discolored with a discoloring solution composed by 40% ethanol and 10% acetic acid (García-Carreño et al., 1993). The clear zones over the blue bottom indicate the protease activity.

Some enzymes need activators, such as Ca2+, DTT and EDTA to show their activity. Those can be added together with the substrate. Other substrates, such as hemoglobin, bovine serum albumin, gelatin and collagen, may have their coloration improved through the use of other dyes, as for example, Amide black (García-Carreño et al., 1993).

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