Quantitative

ENZYMES AND EFFECTS OF pH Thiara Terri V. Bella, Terrence Louis P. Carlos, Joseph Bernard E. Cordova, Jose Alfonso P. Cuisia and Crystel Mhariel V. Daroy Group 2 2F Medical Technology General Biohemistry Laboratory

ABSTRACT Enzymes are biological molecules(proteins) that act as a catalyst and help complex reactions occur everywhere in life. Invertase is an enzyme that catalyzes the breakdown of sucrose to form glucose and fructose. Invertase was extracted from Baker’s yeast and the substrate was collected to determine the effects of changes in pH on the reaction rates of an enzyme-catalyzed reaction. 2.90 ml of buffer was placed in 7 test tubes having a pH of 2,3,4,5,7,9, and 11. The enzyme stock solution was poured and incubated. At the end of the experiment, spectrophotometry was used at 540nm to be able to measure its absorbance.

INTRODUCTION Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For example, they can help break down the foods we eat so the body can use them. Blood clotting is another example of enzymes at work.

sugars and other reducing molecules to form 3amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm[3] thus the need to use spectrophotometry to measure the solution’s absorbance.

Enzymes are needed for all body functions. They are found in every organ and cell in the body, including in the blood, intestinal fluids, mouth(saliva), stomach (gastric juice). [1] Other experimental factors that affect the enzyme activity includes: enzyme concentration, pH of the reaction solution, temperature, substrate concentration, enzyme inhibitors/activators and cofactors/coenzymes. The experiment focused on the effect of pH on the enzyme(invertase) activity. Invertase is an enzyme that catalyzes the hydrolysis (breakdown) of sucrose (table sugar).Alternate names for invertase include EC 3.2.1.26, saccharase, glucosucrase, beta-h-fructosidase, beta-fructosidase, invertin, sucrase, maxinvert L 1000, fructosylinvertase, alkaline invertase, acid invertase and the systematic name: betafructofuranosidase. The resulting mixture of fructose and glucose is called inverted sugar syrup.Invertase is a yeast derived enzyme. It is also synthesized by bees, who use it to make honey from nectar.[2] Denaturation is the complete loss of organized structure in a protein, it is irreversible and it also involves disruption of the secondary and tertiary structure without breaking the primary structure. In the preparation of the solution, invertase was denatured at 95 degrees Celsius to stop activation energy which enabled the group to determine the distinct period. 3,5-Dinitrosalicylic acid (DNS or DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is an aromatic compound that reacts with reducing

Figure 1. Bell shape curve of effects of pH on invertase activity Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH. .

EXPERIMENTAL A. Compounds tested For extraction of invertase from yeast: 0.25 g of Baker’s yeast and 150mL distilled water. For preparation of denatured invertase stock solution: 100mL enzyme stock solution, and boiling water bath.

For effect of pH on invertase activity: 2.90 mL 0.1 M appropriate buffer solution, enzyme stock solution, 60 and 95 degrees Celsius water bath, 3.00mL DNS reagent, 1.50mL sucrose solution and UV-Vis Spectrophotometer. B. Procedure 1. Extraction of Invertase from Yeast 0.25 g of Baker’s yeast was dissolved in 150mL distilled water. It was agitated for 5 minutes was incubated in a water bath with a temperature of 37 degrees Celsius. The solution was decanted and filtered. The supernatant was collected and served as the enzyme stock solution that will be used for the succeeding experiments.

Figure 2. Prepared solutions with different pH

RESULTS AND DISCUSSIONS

2. Preparation of Denatured Invertase Stock Solution 100mL enzyme stock solution was incubated in a boiling water bath for 10 minutes. The solution was allowed to cool and the supernatant can only be collected if frothing or formation of overflowing mass of bubbles occurs. This serves as the denatured enzyme stock solution that will be used for the succeeding experiments. 3. Effect of pH on Invertase Activity 7 numbered test tubes was prepared and 1 test tube for the blank solution. 2.90mL of buffer was poured on every test tube with the pH 2, 3, 4, 5, 7, 9, and 11. 0.10 mL enzyme stock solution was added to each solution, mixed thoroughly, incubated for 60 degrees Celsius water bath for 5 minutes. 1.50mL of sucrose solution was added and incubated again for 5 minutes in a 60 degrees Celsius water bath. 3mL of DNS reagent was added then immersed in a 95 degrees Celsius water bath for 5 minutes to develop the characteristic red-brown color. The solutions was allowed to cool. Blank solution was prepared by following the steps mentioned but the denatured enzyme was used instead of the enzyme stock solution. Absorbance was measured at 540nm. The amount of sucrose hydrolyzed was determined using hydrolyzed-sucrose standard curve constructed in the dinitrosalicylic colorimetric method.

Figure 3. Structures of sucrose and the cyclic (anomeric) conformations of each monosaccharide The figure above shows the activity of invertase as it catalyzes the breakdown of sucrose into glucose and fructose. After the extraction of invertase from the Baker’s yeast, it was used to determine the effects of pH on the enyme activity. In this experiment, the rate of reaction is based on the amount of sucrose hydrolyzed by the invertase. Consequently, the reaction rate usually increases as the amount of sucrose hydrolyzed by the invertase . Thus, the reaction rate has a direct relationship to the amount of sucrose hydrolyzed. Invertase splits the disaccharide sucrose into the monosaccharides glucose and fructose. Invertase is inhibited by high concentrations of its substrate, sucrose. The invertase we supply has optimum activity at 60 °C. Its optimum pH is 4.5 (the pH is usually adjusted to this level by the addition of citric acid to the reaction mix), although it is active between pH 3.0 and 5.5. Inactivation of the enzyme begins at 65 °C and the enzyme is totally inactivated after 5 minutes at 90 °C.

direct relationship with the amount of sucrose hydrolyzed(mg/mL). Table 3 would yield a chart as seen in Figure 5 below. Figure 5. Graph of pH vs. Absorbancej

Figure 4. Standard curve plotted from the Colorimetric method and the amount of sucrose hydrolyzed per minute as a function of pH In the sucrose assay using Dinitrosalicylic Colorimetric method, absorbance was compared to the amount of acid-hydrolyzed sucrose which produced a standard curve that as absorbance increases, the amount of hydrolyzed sucrose also increases. As seen also in figure 4, the amount of hydrolyzed sucrose was highest at around pH 45. Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability.This is the pH wherein the enzyme invertase is most active or what we call as the optimum pH Table 3. Effect of pH on Invertase Activity pH 2 3 4

Absorbance at 540nm -0.004A -0.012A -0.011A

5 7 9 11

0.011A -0.008A 0.004A -0.002A

Sugar Assay using dinitrosalicylic colorimetric method was not done in the experiment and absorbance was used as a reference to know the optimum pH of the invertase. Absorbance at 540nm can be used as a reference since it has a

As seen in the graph, the highest peak is at pH4-5 which means this the optimum pH wherein the enzyme invertase is most active hydrolyzing also most of the sucrose. We can’t use equimolar concentrations of glucose and sucrose as a standard solution for the construction of standard curve for it would not yield an upward slope graph but instead the opposite. Some errors were made during the experiment so the bell shaped curve which is the correct curve of the graph was not achieved. The curve doesn’t show a definite relationship between pH and the rate of invertase activity for the invertase activity is only at its peak at optimum pH. In addition to temperature and pH there are other factors, such as ionic strength, which can affect the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimized in order for an enzymatic reaction to be accurate and reproducible.

REFERENCE: [1] Retrieved from http://www.worthingtonbiochem.com/introbiochem/effectsph.html on Mar. 17, 2015 [2] Retrived from http://cdn.intechopen.com/pdfswm/26595.pdf on Mar. 17, 2015

[3] Retrieved from http://www.nlm.nih.gov/medlineplus/ency/ar ticle/002353.htm on Mar. 17,2015 [4] Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 31(3), 426-428 [5] Wang, N.S. Experiment 14: Enzyme kinetics of invertase via initial rate determination.Retrieved from http://www.eng.umd.edu on Mar. 17,2015 [6] Wang, N.S. Experiment 9D: Sucrose assay by dinitrosalicylic acid colorimetric method.Retrieved from http://www.eng.umd.edu on Mar. 17,2015