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University of Santo Tomas Faculty of Pharmacy Biochemistry Laboratory

DETERMINATION OF INVERTASE ACTIVITY BY DINITROSALICYLIC COLORIMETRIC METHOD AND EFFECTS OF pH
Santia, A.; Salandanan, R.; Santos, C.; Santos, E.; Sibayan, Q.; Supillo, A. Group 7 2B Pharmacy Biochemistry Laboratory

The activity of enzyme is affected by different factors and one of which is the pH. The point where the enzyme exhibits its maximum activity is known as the optimum pH. In this experiment, the invertase was extracted from the yeast and used as the experimental enzyme. Four different pH levels of buffered solution were used specifically: pH 3, 5, 7, and 10. Negative values were observed from the UV-Vis Spectrophotometer. The amount of sucrose hydrolyzed was also calculated applying the equation obtained from the sucrose assay using Dinitrosalicylic Colorimetric method. A bell-shaped graph resulted from the values gathered and pH 5 was observed to be the optimum pH of the enzyme invertase.

ABSTRACT

INTRODUCTION

Enzymes are usually globular proteins that act as biological catalysts responsible for speeding up the rate of a chemical reaction. In an enzyme-catalyzed reaction, the enzyme binds to the substrate, which is one of the reactants, to form a complex. The formation of the complex leads to the formation of the transition-state species, which then forms the product [1]. The enzymes work by reducing the activation energy that is essential for starting a reaction. Thus by lowering the activation energy, the rate of reaction is increased. The activity of enzymes depends on various factors, such as the effect of enzyme concentration, pH, and temperature. This experiment focuses on the effect of pH on enzymatic activity. The measure of the concentration of hydrogen ions in a solution is known as the pH. Since enzymes are proteins, they are very responsive to changes in pH. They usually function over a narrow range of pH. Thus, changes in pH affect the rate of the enzyme reaction significantly. Denaturation, or loss of activity of protein occurs at extremely low or high levels of pH [2] . Each enzyme has its own optimum pH value, in which the maximum activity is achieved. The graph of the rates of most enzyme-catalyzed reactions exhibits a bellshaped curve as shown on the right side.

Figure 1. Effect of pH on reaction rate The bell-shaped curve indicates the stability of enzymes during the alteration of pH. Hence, extremely low and high pH results to slow reaction rate because of loss of enzymatic activity. The peak of the curve reveals the best or optimum pH suitable for the enzyme because it reaches the maximum reaction rate of the enzymatic activity. Dinitrosalicylic colorimetric method is a test for the presence of free carbonyl group (C=O), or the so-called reducing sugars [3]. This method involves the oxidation of the aldehyde functional group present in glucose. The reagent used is a yellow dye known as 3,5-dinitrosalicylic acid (DNS), which is primarily used to determine the sugar content. At the same time, DNS is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions. Aside from the oxidation of the carbonyl groups in the sugar, the decomposition of sugar also competes for the availability of DNS. For this reason, carboxymethyl

50 mL distilled water 1. they were incubated for 5 minutes in a 60oC water bath.cellulose can affect the calibration curve by enhancing the intensity of the developed color. Table 1. Three drops of concentrated HCl was added to each test tube. Test Tube Preparation for Sucrose Assay Using DNS Colorimetric Method Test Tube No.2. B. One and a half mL of sucrose solution was added and the test tubes were again incubated at the same temperature for another 5 minutes. This is because of the conversion of the 3. Sucrose Assay by Dinitrosalicylic Colorimetric Method In the experiment. Then.50 0. The absorbance was measured at 540 nm with the use of the UV-Vis Spectrophotometer. a product from invertase activity of sucrose. Samples Used Standard sucrose solution was used for the sucrose assay determination while invertase stock and denatured solutions that were extracted from yeast were used for the effects of pH. Three mL of DNS reagent was placed in the mixture. The solution was mixed and incubated at 90oC water bath for 5 minutes.1 mL was added to each test tube.5 M KOH.1. Blank solutions were prepared by following the previous steps but instead of enzyme stock solution. The DNS reagent with the amount of 3 mL was then added and the test tubes were immersed in 95oC water bath to develop the characteristic redbrown color. the following values were obtained (Table 3) and the standard curve was generated together with the equation through the use of MS Excel (Figure 3). Effect of pH on Invertase Activity Four numbered test tubes were prepared and 2. After the solutions were mixed.1 M buffer solution was added afterwards. pH 1 3 2 5 3 7 4 10 Figure 2. Procedure B.90 mL 0. Test Tube Preparation for Effect of pH on Invertase Activity Test Tube No.25 0. .15 mL 0. the test tubes were cooled and their absorbance was measured at 540 nm.25 1. the content was neutralized by adding 0.75 0. Subsequently.25 0 An enzyme stock solution with amount of 0. The DNS also reacted to glucose. denatured enzyme solution was used. Sucrose Assay Colorimetric Method by Dinitrosalicylic A series of test tubes were prepared in accordance to the table found below (Table 1). solution 0 0.5-dinitrosalicylic acid EXPERIMENTAL A.8 mL of 0. B.50 0.00 0. the solution slowly turned to a red-brown color. About 2.75 1. and converted to gluconic acid. The test tubes were cooled afterwards.25 1. RESULTS AND DISCUSSION A. Blank 1 2 3 4 5 6 mL sucrose std.50 1. By measuring the absorbance using UVVis Spectrophotometer.1 M buffer solution with different pH levels was added in each test tube: Table 2. The test tubes were then immersed in 95oC water bath for 10 minutes to develop the characteristic red-brown color. Structure of 3. after the DNS was incorporated in the test tubes with the presence of heat.5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid.00 1.

049 0. and from the computed data in Table 5. a linear trend was identified as well as the slope-intercept form that was y=0. the optimum pH was determined.33 6.023 0. the slope-intercept form obtained was y=0. Absorbance vs. Amount of AcidHydrolyzed Sucrose In the graph shown above.842 mg/mL Table 5.0242 while the slope was 0.038 10 -0. Table 4.316 mg/mL At pH 5: x=(-0.022 5 -0. Absorbance values (540 nm) at certain pH level pH Absorbance at 540nm 3 -0.67 20 Absorbance at 540nm 0.737 mg/mL At pH 7: x=(-0. the highest would be the value closest to the origin.0242 0.316 -22.737 -34.019)–0. a formula was derived from the slopeintercept form.0462 0.038)–0.0019 x= -22.0242 0.0019 x= -24.019 7 -0.0242. B.842 Figure 3.737 mg/mL At pH 10: x=(-0. From the values in Table 4.Table 3.0019x+0.33 16. .022)–0.04 0. Amount of Acid-Hydrolyzed Sucrose at certain pH level pH 3 5 7 10 Amount of AcidHydrolyzed Sucrose -24. y=mx+b Derived formula: x=y – b m y=absorbance b=intercept m=slope x= concentration or amount of acidhydrolyzed sucrose The intercept from the given equation was 0.0019x+0. The optimum pH value exhibits the maximum enzymatic activity which means it is the pH with the highest amount of concentration achieved from the enzyme-catalyzed reaction.737.0242.019 under pH 5.0019. Since the obtained values were all negative values. Values for Amount of AcidHydrolyzed Sucrose and Absorbance at 540 nm using DNS method Concentration 0 3.0242 0.03 0. the highest absorbance was -0.042)–0.67 10 13.042 At pH 3: x=(-0. The following absorbance values obtained from the spectrophotometer will be used to compute Based from the results.0019 x= -34.0019 x= -32.737 -32.0242 0. Effect of pH on Invertase Activity From the sucrose assay standard curve constructed. In order to get the concentration (x).046 0.067 for the concentration or amount of acidhydrolyzed sucrose. pH 5 also has the highest amount of acidhydrolyzed sucrose which is -22.

Use of this enzyme at pH values extremely lower or higher than the range is not suggested because it causes denaturation or loss of activity of the enzyme. Biochemistry. From the internet Retrieved January 14. California: . (1995).html Figure 4. 2012. from http://www. pH The experimental graph shows a nearly bell-shaped curve. Hence.eng. New York: Van Nostrand Reinhold.com/introbiochem/effectsph. S. Nevertheless. which should have been obtained if another low pH value was used. New Jersey: Prentice Hall.html Retrieved January 14. Amount of Acid-Hydrolyzed Sucrose vs. and 7th Farrell.html Retrieved January 14. Understanding Enzymes. (2009).ausetute. S.worthingtonbiochem. 2012. from http://www. the optimum pH was determined and it is pH 5. 2nd ed. as observed in the given tables and in the figure above. Reiner. Palmer. Retrieved January 14. the invertase enzyme may be used over an extended pH range with an optimum pH of 5. which illustrated the expected bell-shaped curve of the reaction.com.au/enzymes.chemguide. 2012. from http://www.html REFERENCES [1] Campbell. since there were only four pH levels used in the experiment. 2012.umd. M. It lacks the first part of the curve. ed. 4th ed. Brooks/Cole. and Farrell.uk/organicprops /aminoacids/enzymes2. (2009) [2] Von Euler and colleagues (1924) and Dixon (1953) [3] Miller (1959) From books Campbell. M. T. Behavior of Enzyme Systems.edu/~nsw/ench485/l ab4a. J.Found below is the graph of the values from Table 5.co. from http://www. (1969).