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DETERMINATION OF ENZYMATIC ACTIVITY BY DINITROSALICYLIC COLORIMETRIC METHOD AND pH Esclanda, Verna Mae F., Flores, Mary Camelle C.

, Galvez, Czarina M., Gamboa, Maureen Allysandra J., Go Imon, Karl Louis L., Gudio, Klaudine Renee T. ABSTRACT

Enzyme activity is affected by several factors. When the enzyme changes in shape and structure, its rate of reaction is varied. Examples of the things that alters the rate of reaction of the enzymatic activity are pH and addition of chemicals or reagents. In this experiment, dinitrosalicylic acid colorimetric method and changing in pH were done to determine the effects of physical and chemical factors to the invertase activity of sucrose. Negative absorbance were observed after they were tested in the UV-Vis Spectrophotometer.

INTRODUCTION
Enzymes are proteinaceous catalysts, which speed up the rate of a biochemical reaction. They reduce the activation energy that is essential for starting any type of chemical reaction. With a low energy requirement for activation, the reaction takes place faster. The overall performance of an enzyme depends on various factors, such as temperature, pH, cofactors, activators and inhibitors.1 Since enzymes are proteins, they are very sensitive to changes in pH. Each enzyme has its own optimum range for pH where it will be most active. This is the result of the effect of pH on a combination of factors: (1) the binding of the enzyme to substrate, (2) the catalytic activity of the enzyme, (3) the ionization of the substrate, and (4) the variation of protein structure.2 The initial rates for many enzymatic reactions exhibit bell-shaped curves as a function of pH as shown in the example below.

enzyme that it reaches the maximum reaction rate of the enzymatic activity. Dinitrosalicylic acid (D.N.S.A. or 3:5dinitrosalicylic acid) is a (yellow) reagent used to determine sugar content especially glucose. The DNS technique is employed in order to estimate sugar present in the blood, in the cerebrospinal fluid and in other human bodily fluids. This is also effectively used in the handling of requirements for diabetic clinics in hospital laboratories, considering that only 10 minutes is enough for the process to take place and the reagents are stable, cheap and easily prepared. The amount of blood sugar in the blood has metabolic implications and is used to determine the presence of blood sugar-related disorders such as hyperglycemia. One good way to assess blood sugar level is through the use of dinitrosalicylic acid.3

Fig.2 Chemical structure of 3,5-dinitrosalicylic acid Fig.1 Effect of pH on enzymatic activity

The best explanation for this bell-shaped curve is the stability of enzymes during the alteration of pH. Meaning, low pH results to slow reaction rate but too high pH shows the same result because of enzymatic loss. The peak of the bell demonstrates the best pH suitable for the

This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5dinitrosalicylic acid (DNS) is reduced to 3-

amino,5-nitrosalicylic conditions:

acid

under

alkaline

the calibration curve by enhancing the intensity of the developed color.4

METHODOLOGY

Because dissolved oxygen can interfere with glucose oxidation, sulfite, which itself is not necessary for the color reaction, is added in the reagent to absorb the dissolved oxygen. The above reaction scheme shows that one mole of sugar will react with one mole of 3,5dinitrosalicylic acid. However, it is suspected that there are many side reactions, and the actual reaction stoichiometry is more complicated than that previously described. The type of side reaction depends on the exact nature of the reducing sugars. Different reducing sugars generally yield different color intensities; thus, it is necessary to calibrate for each sugar. In addition to the oxidation of the carbonyl groups in the sugar, other side reactions such as the decomposition of sugar also competes for the availability of 3,5-dinitrosalicylic acid. As a consequence, carboxymethyl cellulose can affect

A. Sucrose Assay By Dinitrosalicylic Colorimetric Method A series of test tubes were prepared as seen in the table below (Table 1). Three drops of concentrated HCl was added to each tube. The tube was mixed then incubated in a 90oC water bath for 5 minutes. Then, 0.15 ml 0.5 M KOH was added to neutralize the solution. A 0.1 M buffer solution about 2.8 ml was then added and mixed. Three ml of DNS reagent was put in the mixture. The test tubes were afterward immersed in 95oC water bath for 10 minutes to develop the characteristic red-brown color. The test tubes were then cooled and their absorbance were measured at 540 nm.

Table 1. Test Tube Preparation For Sucrose Assay Using Dinitrosalicylic Colorimetric Method

Test Tube No./ Volume Sucrose Std. Solution (1mg/ml) Distilled Water

Blank 0 ml 1.50 ml

1 0.25 ml 1.25 ml

2 0.50 ml 1.00 ml

3 0.75 ml 0.75 ml

4 1.00 ml 0.50 ml

5 1.25 ml 0.25 ml

6 1.50 ml 0 ml

B. Effect of pH on Invertase Activity Six numbered test tubes were prepared. 2.9 ml 0.1 M buffer solution with different pH was added in each test tube:
Table2. Test Tube Preparation For Effect of pH on Invertase Activity

Test Tube No. pH

1 2

2 3

3 7

4 7.5

5 11

for another 5 minutes. Three ml of DNS reagent was then added. The test tubes were immersed in 95oC water bath to develop the characteristic red-brown color. The test tubes were cooled after. Blank solutions were prepared by following the first steps but instead of enzyme stock solution, denatured enzyme solution was added. The absorbance was measured at 540 nm.

Enzyme stock solution with amount of 0.1 ml was added to each test tube. After the test tubes were mixed, they were incubated in 60oC water bath for 5 minutes. One and a half ml of sucrose solution was added and the test tubes were again incubated in 60oC water bath

RESULTS AND DISCUSSION


A. Sucrose Assay Using Dinitrosalicylic Colorimetric Method In the experiment, as the dinitrosalicylic acid, a yellow dye, was incorporated in the test tubes with the presence of heat, the mixture slowly turned red-brown. This is because the conversion of the 3,5dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, which contributed to the red-brown color of the mixture. The DNS also reacted to glucose, a product from invertase activity of sucrose, and converted to gluconic acid. The absorbance of each hydrolyzed sucrose on test tubes was identified using the UV-vis spectrophotometer.

Table3. x and y Values for Amount of Acid-Hydrolyzed Sucrose and Absorbance at 540nm

Amount of Acid-Hydrolyzed Sucrose (x) 0 0.017 0.033 0.050 0.067 0.250 0.100

Absorbance at 540nm (y) 0 0.004 -0.001 -0.003 -0.003 0.048 0.001

Fig.3 Breakdown of Sucrose to Glucose and Fructose Through Invertase

To compute for the amount of acidhydrolyzed sucrose, the equation was used. Using the formula, the following were obtained:

Fig.4 Absorbance vs. Amount of Acid-Hydrolyzed Sucrose

The line drawn on the graph represented the best fit line and was computed through the linear regression function of a scientific calculator. The slope-intercept form computed was found to be y=0.215x-0.011. In the graph shown for absorbance vs. amount of acid hydrolyzed sucrose, a linear trend was not identified. This is due to some possible causes. First, inactivity of the sucrose if it was not freshly prepared. Second, the dinitrosalicylic acid might not be reactive. Or, the spectrophotometer was defective or not sensitive enough and may actually commit errors. B. Effect of pH on Invertase Activity

y=absorbance b=intercept m=slope

The intercept was found to be -0.011 while the slope was 0.215. The following absorbance will be used to compute for the concentration or amount of acid-hydrolyzed sucrose:
Table4. Absorbance (at 540 nm) at certain pH

Invertase may be used over an extended pH range with an optimum pH at 4.5. This enzyme is fully active from pH 3.0 to 5.5. Use at pH values over 6.0 is not recommended because it causes denaturation of the enzyme. The result of the experimental graph was very far different from the ideal graph. The graph was supposed to be bell shaped. But in this experiment, the graph was U-shaped. This Ushaped curve was due to the negative values of the absorbance. This kind of error was committed because of some possible factors like the pH of the mixture was not accurate or the spectrophotometer itself committed the errors.

pH 2 3 7 7.5 11

Absorbance at 540nm 0.036 -0.004 -0.013 -0.009 0.009

REFERENCES
1

PH EFFECT ON ENZYMES. (n.d.). Retrieved January 18, 2011, from http://www.buzzle.com/articles/ph-effect-onenzymes.html 2 ENZYME ACTIVITY. (n.d.). Retrieved January 18, 2011, from http://www.rpi.edu/dept/chem-eng/BiotechEnviron/IMMOB/enzymeac.htm 3 Tan, Sue Teresa. HOW TO USE DINITROSALICYLIC ACID Retrieved January 18, 2011 http://www.ehow.com/how_5221277_usedinitrosalicylic-acid.html#ixzz1BIa2r4fx
Table5. x and y Values for Effect of pH on Invertase Activity
4

pH (x) 2 3 7 7.5 11

Amount of Acid-Hydrolyzed Sucrose (y) 0.218 0.033 -0.0093 0.0093 0.09

Wang, N.S. GLUCOSE ASSAY BY DINITROSALICYLIC COLORIMETRIC METHOD Retrieved January 18, 2011, from

http://www.eng.umd.edu/~nsw/ench485/lab4 a.htm

Fig.5 Amount of Acid-Hydrolyzed Sucrose vs. pH