University of Santo TomasFaculty 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

ABSTRACTThe 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.

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 bell-shaped 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 cellulose can affect the calibration curve by enhancing the intensity of the developed color.

Figure 2. Structure of 3,5-dinitrosalicylic acid

EXPERIMENTALA. 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.

B. Procedure B.1. Sucrose Assay by Dinitrosalicylic Colorimetric Method A series of test tubes were prepared in accordance to the table found below (Table 1). Three drops of concentrated HCl was added to each test tube. The solution was mixed and incubated at 90oC water bath for 5 minutes. Then, the content was neutralized by adding 0.15 mL 0.5 M KOH. About 2.8 mL of 0.1 M buffer solution was added afterwards. Three mL of DNS reagent was placed in the mixture. The test tubes were then immersed in 95oC water bath for 10 minutes to develop the characteristic red-brown color. Subsequently, the test tubes were cooled and their absorbance was measured at 540 nm.

Table 1. Test Tube Preparation for Sucrose Assay Using DNS Colorimetric Method

B.2. Effect of pH on Invertase Activity Four numbered test tubes were prepared and 2.90 mL 0.1 M buffer solution with different pH levels was added in each test tube:

Table 2. Test Tube Preparation for Effect of pH on Invertase Activity

An enzyme stock solution with amount of 0.1 mL was added to each test tube. After the solutions were mixed, they were incubated for 5 minutes in a 60oC water bath. 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. 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 red-brown color. The test tubes were cooled afterwards. Blank solutions were prepared by following the previous steps but instead of enzyme stock solution, denatured enzyme solution was used. The absorbance was measured at 540 nm with the use of the UV-Vis Spectrophotometer.

RESULTS AND DISCUSSIONA. Sucrose Assay by Dinitrosalicylic Colorimetric Method In the experiment, after the DNS was incorporated in the test tubes with the presence of heat, the solution slowly turned to a red-brown color. This is because of the conversion of the 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid. The DNS also reacted to glucose, a product from invertase activity of sucrose, and converted to gluconic acid. By measuring the absorbance using UV-Vis Spectrophotometer, 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).

Table 3. Values for Amount of Acid-Hydrolyzed Sucrose and Absorbance at 540

nm using DNS method

Figure 3. Absorbance vs. Amount of Acid-Hydrolyzed Sucrose

In the graph shown above, a linear trend was identified as well as the slope-intercept form that was y=0.0019x+0.0242.

B. Effect of pH on Invertase Activity From the sucrose assay standard curve constructed, the slope-intercept form obtained was y=0.0019x+0.0242.

y=absorbance b=intercept

Derived formula: m=slope x= concentration or amount of acid- hydrolyzed sucrose

The intercept from the given equation was 0.0242 while the slope was 0.0019. In order to get the concentration (x), a formula was derived from the slope-intercept form. The following absorbance values obtained from the spectrophotometer will be used to compute for the concentration or amount of acid-hydrolyzed sucrose.

Test TubeNo.

mL sucrose std. solution

mL distilled water

Blank 0 1.50

1 0.25 1.25

2 0.50 1.00

3 0.75 0.75

4 1.00 0.50

5 1.25 0.25

6 1.50 0

Test TubeNo.

1 2 3 4

pH 3 5 7 10

Concentration Absorbance at 540nm0 0.023

3.33 0.036.67 0.0410 0.046

13.33 0.046216.67 0.049

20 0.067

y=mx+b

x=y – b m

Table 4. Absorbance values (540 nm) at certain pH level

pH Absorbance at 540nm3 -0.0225 -0.0197 -0.038

10 -0.042

At pH 3:

At pH 5:

At pH 7:

At pH 10:

Table 5. Amount of Acid-Hydrolyzed Sucrose at certain pH level

Based from the results, the optimum pH was determined. 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. Since the obtained values were all negative values, the highest would be the value closest to the origin. From the values in Table 4, the highest absorbance was -0.019 under pH 5, and from the computed data in Table 5, pH 5 also has the highest amount of acid-hydrolyzed sucrose which is -22.737. Found below is the graph of the values from Table 5, which illustrated the expected bell-shaped curve of the reaction.

Figure 4. Amount of Acid-Hydrolyzed Sucrose vs. pH

The experimental graph shows a nearly bell-shaped curve, since there were only four pH levels used in the experiment. It lacks the first part of the curve, which should have been obtained if another low pH value was used. Nevertheless, the optimum pH was determined and it is pH 5, as observed in the given tables and in the figure above. Hence, the invertase enzyme may be used over an extended pH range with an optimum pH of 5. 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.

REFERENCES[1] Campbell, M. and Farrell, S. (2009)[2] Von Euler and colleagues (1924) and Dixon (1953)[3] Miller (1959)

From books

Campbell, M. and Farrell, S. (2009). Biochemistry. 7th ed. California: Brooks/Cole.

Palmer, T. (1995). Understanding Enzymes. 4th ed. New Jersey: Prentice Hall.

Reiner, J. (1969). Behavior of Enzyme Systems. 2nd ed. New York: Van Nostrand Reinhold.From the internetRetrieved January 14, 2012, from http://www.ausetute.com.au/enzymes.html

Retrieved January 14, 2012, from http://www.chemguide.co.uk/organicprops/aminoacids/enzymes2.html

pH Amount of Acid-Hydrolyzed Sucrose

3 -24.3165 -22.7377 -32.737

10 -34.842

x=(-0.022)–0.0242 0.0019

x= -24.316 mg/mL

x=(-0.019)–0.0242 0.0019

x= -22.737 mg/mL

x=(-0.038)–0.0242 0.0019

x= -32.737 mg/mL

x=(-0.042)–0.0242 0.0019

x= -34.842 mg/mL

Retrieved January 14, 2012, from http://www.eng.umd.edu/~nsw/ench485/lab4a.html

Retrieved January 14, 2012, from http://www.worthington-biochem.com/introbiochem/effectsph.html