EFFECTS OF CHANGES IN TEMPERATURE ON REACTION RATES OF AN ENZYME-CATALYZED REACTION

Sandimas, Gabriel Jerome., Semillano, Charisse., Sta. Ana, Mariel P., Tiad, Erwin C., Timbol, Ida Maribeth G.

Group 9 2GPH Biochemistry Laboratory

ABSTRACT

Several factors affect the rate at which enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators. In this experiment, dinitrosalicylic colorimetric method and changes in temperature in a sucrose solution were done in order to determine the chemical and physical effects on the invertase activity.

INTRODUCTION

Chemically, enzymes are generally globular proteins. They speed up the rate of chemical reactions because they lower the energy of activation, the energy that must be supplied in order for molecules to react with one another. Enzymes lower the energy of activation by forming an enzyme-substrate complex allowing products of the enzyme reaction to be formed and released. Enzyme activity is affected by a number of factors including; the concentration of enzyme, substrate and salt and the temperature and pH. [1]

As for their sensitivity to temperature changes, like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. This has several effects on the rates of reactions; a.) more energetic collisions, b.) the number of collisions per unit time will increase, c.) the heat of the molecules in the system will increase. [2]

As shown in Figure 1, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. This is called the optimum temperature at which it works best.

This bell-shaped curve depicts the stability of the enzymes during changes in temperature. Meaning, a low temperature slows the reaction just like a temperature which is too high has the same result because of enzymatic loss. The peak of the bell demonstrates the best temperature suitable for the enzyme and would mean that the maximum reaction rate of the enzyme has been reached.

3,5-Dinitrosalicylic acid (DNS or DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic

acid, which absorbs light strongly at 540 nm. It was first introduced as a method to detect reducing substances in urine and has since been widely used, for example, for quantification of carbohydrates levels in blood. It is mainly used in assay of alpha-amylase. However, enzymatic methods are usually preferred to DNS due to their specificity. [3]

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. DNS reacts with the sugar to form 3-amino, 5-nitrosalicylic acid in a reduction reaction. In other words, one mole of sugar will react with 1 mole of DNS to form 3-amino, 5-nitrosalicylic acid. The chemical that is formed is able to absorb light strongly at 540 nm and depending on the nature of the sugars, the darker the reaction will be. So the more sugar there is, the more of carbonyl groups there are, and the darker it will be (copper red color).

The reaction is shown below:

      oxidation       aldehyde group carboxyl group                    reduction              3,5-dinitrosalicylic acid 3-amino,5-nitrosalicylic acid

Heating the solution liberates the reducing sugars which allow the 3, 5-dinitrosalicylic acid to react with the reducing sugar and in turn, producing the color. [4]

EXPERIMENTAL

A. SUCROSE ASSAY USING DINITROSALICYLIC COLORIMETRIC METHODA series of test tubes covered with marbles were prepared as follows:

Tube no. Blank 1 2 3 4 5 6mL sucrose standard solution

0 0.25 0.50 0.75 1.00 1.25 1.50

mL distilled water

1.50 1.25 1.00 0.75 0.50 0.25 0

Table 1Test tube preparation of Sucrose Assay Using Dinitrosalicylic Colorimetric Method

Afterwhich, 3 drops (0.05 mL) concentrated HCl were added to each test tube. This were individually mixed and incubated at 90 C water bath for 5 minutes. An addition of 0.15 mL of 0.5 M KOH to neutralize the solution followed. Then, 2.80 mL of 0.1 M buffer solution, pH 5 was also added and mixed well. Finally, 3 mL of DNS reagent was added in each of the test tubes before they were immersed in 95 C water bath for 10 minutes at which they developed the characteristic red-brown color.

Figure 3. Test tubes upon addition of DNS reagent

Upon cooling the absorbance was measured at 540 nm. As the final step, the hydrolyzed-sucrose standard curve was constructed by plotting A540 against its concentration (mg/mL).

B. EFFECT OF TEMPERATURE ON INVERTASE ACTIVITY

Initially, 20, 30, 50, 60, 70 and 90 C water baths were set up. Then, 6 test tubes each containing 1.5 mL sucrose solution were prepared and incubated separately for 5 minutes in each water bath. In another test tube, 0.80 mL of enzyme stock solution was mixed with 19.20 mL of 0.1 buffer solution, pH 5. An addition of 3 mL of dilute enzyme

solution was added to all test tubes before they were incubated for another 5 minutes without being removed from their respective water baths. After incubation 3 mL of the DNS reagent was added. The test tubes were then immersed in a 95 C water bath for 10 minutes for the characteristic red-brown color to develop.

Figure 4. Immersion of the test tubes in a 95 C water bath

The solutions were then allowed to cool. Again, marbles were used to cover them in order to prevent the evaporation of the solvent. In a different set of 6 test tubes the procedure was repeated only denatured enzyme was added instead of enzyme stock solution. The absorbance of which was measured at 540 nm.

Figure 5. Solutions were placed in different cuvettes prior to spectrophotometry

Finally, the amount of sucrose hydrolyzed was likewise determined using the sucrose standard curve constructed in the dinitrosalicylic colorimetric method done earlier.

Figure 6. Spectrophotometer to determine the light absorbance at 540nm