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Romina, Cyla Mariel C. Salvadora, Joanna Gee L.H. San Pedro, Gabrielle Luis B. Santos Goup 6 2F Pharmacy Biochemistry Laboratory ABSTRACT
Enzyme activity is affected by different factors like temperature, pressure, chemical environment or pH levels, and the concentration of substrate. When the enzyme changes in shape and structure, its rate of reaction varies. In this experiment, invertase was extracted from Bakers yeast and was subjected to dinitrosalicylic (DNS) colorimetric method and changes in pH to determine the effects of physical and chemical factors to the invertase activity of sucrose. Through graphical representation, points were plotted to determine the best-fit line and the curve of the graph.

INTRODUCTION Enzymes are protein molecules which can be defined as biological catalysts. A catalyst is a molecule which speeds up a chemical reaction, but remain unchanged at the end of the reaction. Enzymes are globular proteins which means that their molecules are coiled into a precise 3D shape with hydrophilic R groups on the outside of the molecule ensuring that they are soluble. [1] Invertase is a yeast derived enzyme that can generally cleave peptide bonds and specifically hydrolyze sucrose to glucose and fructose. The official name for invertase is betafructofuranosidase (EC3.2.1.26), which implies that the reaction catalyzed by this enzyme is the hydrolysis of the terminal nonreducing betafructofuranoside residues in betafructofuranosides. [2] Sucrose, commonly known as table sugar, is a disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,4-glycosidic bond. When this bond is cleaved in a hydrolysis reaction, an equimolar mixture of glucose and fructose is generated. This mixture of monosaccharides is called invert sugar, which is derived from the fact that sucroserotates plane polarized light to the right whereas the hydrolysis products rotates plane polarized light to the left. [3] Glucose is by far the most common carbohydrate and classified as a monosaccharide and is a reducing sugar. It is also known as dextrose and blood sugar as it circulates in the. It is initially synthesized by chlorophyll in plants using carbon dioxide from the air and sunlight as an energy source. Glucose is further converted to starch for storage.[4] Dinitrosalicylic acid (DNS) is a yellow reagent used to determine sugar content specifically glucose. DNS Assay is the method utilized to monitor enzymatic activity of invertase.

Spectrophotometer was used to determine the absorbance of the solution to be able to plot the graph for the different pH. EXPERIMENTAL A. Compounds Usedbakers yeast, enzyme stock solution, denatured enzyme stock solution B. Samples Usedsucrose solution, glucose, 3,5-Dinitrosalicyclic, pH (2, 3, 5, 8, 9, and 11), 0.5M KOH, Concentrated HCl C. Procedure [5] For the extraction/isolation of invertase from yeast, 0.25g of Bakers yeast was dissolved in distilled water to make a 250mL solution. The solution was allowed to stand for 20 minutes and the supernatant was collected. The supernatant served as the enzyme stock solution (ESS). Test Tube mL sucrose solution mL distilled water Bla -nk 0 3 1 0.5 2.5 2 1 2 3 1.5 1.5 4 2 1 5 2.5 0.5 6 3 0

Table 1. Test tube preparation for glucose assay

In the glucose assay using DNS colorimetric method, 7 numbered test tubes were prepared with the contents from table 1 and was added 3 drops of concentrated HCl. This was incubated at 90C water bath for 5 minutes. The solution was neutralized by adding 0.15mL 0.5M KOH and was added 2.80mL 0.1 buffer solution (pH5). Three mL of DNS reagent was added and the test tube was immersed in 95C water bath for 10

minutes to develop red-brown color. After cooling, the absorbance was measured. In the test for the effect of pH on invertase activity, six numbered test tubes each contained 2.9mL of different pH (2,3, 5, 8, 9, and 11) and was added 0.1mL of ESS.

RESULTS AND DISCUSSION In the glucose assay using DNS colorimetric method, when DNS was incorporated in the solution in the presence of heat, it was observed that the solution slowly turned red-brown. This is because the conversion of the 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid. The absorbance of each hydrolyzed sucrose on test tubes was identified using the UV-vis spectrophotometer. Test tube number Blank 1 2 3 4 5 6 Amount of Glucose Formed (mg/mL) 0 5.56x10-3 0.01 0.016 0.022 0.027 0.033 Absorbance540nm

0 0.207 0.285 2.540 2.750

Figure 1.1 Incubation of ESS and pH

This was incubated in 60C water bath for 5 minutes. The test tube was taken out and was added with 1.5mL of sucrose solution and was again incubated in 60C water bath for 5 minutes.

Table 2. Glucose Assay Using DNS Colorimetric Method

The line drawn on its graphical representation is the best fit line and was computed through the linear regression function of a scientific calculator. The slope was found to be 105.9 while the intercept was -0.42. The enzyme invertase was extracted from Bakers yeast was used as the enzyme stock solution. This was then added to different pH to test the effects of pH to enzymatic activity. Table 2 shows the amount of glucose formed (in mg/mL) and absorbance540nm while figure2 shows the graphical representation of pH and absorbance. pH Amount of Glucose Formed (mg/mL) 2.71x10-3 2.57x10-3 2.41x10-3 3.50x10-3 3.96x10-3 2.95x10-3 Absorbance540nm 0.044 0.030 0.015 0.129 0.166 0.065

Figure 1.2 Incubation of ESS, pH, glucose, and DNS

The test tube was again taken out and 3.0mL of DNS was added and the test tube was immersed in 95C for 10 minutes to develop the characteristic red-brown color. A blank solution was prepared for each pH following the same procedure but instead of adding 0.1mL of ESS, 0.1mL of denatured ESS was added. The absorbance was measured using the spectrophotometer at 540nm. A graphical representation of pH and absorbance was plotted.

2 3 5 8 9 11

Table3. Effect of pH on Invertase Activity

To compute for the amount of glucose formed (mg/mL), the equation ; where in y is the absorbance, b is the intercept which is -0.42, and m is the slope which is 105.9

pH 2: X= 2.71x10-3mg/mL pH 3: X= 2.57x10-3mg/mL pH 5: X=2.41x10-3mg/mL pH 8: X= 3.50x10-3mg/mL pH 9:

X=3.96x10 mg/mL

[5] Crisostomo, A.C., et al. Laboratory Manual in General Biochemistry (2010). Pp 42-43. C & E Publishing House. Philippines: Quezon City.

pH 11: X=2.95x10-3mg/mL 0.2 0.15 0.1 0.05 0 0 2 4 6 8 10 12

Figure2. Effect of pH on Invertase Activity

The graph shows a bell-shaped curve because it shows that there is an optimum pH at which the enzyme is active maximally. REFERENCES [1] http://www.nlm.nih.gov/medlineplus/ency /article/002353.htm [2] http://greenwoodhealth.net/np/invertase. htm [3] http://www.elmhurst.edu/~chm/vchembo ok/546sucrose.html [4] http://www.elmhurst.edu/~chm/vchembo ok/543glucose.html