EFFECT OF TEMPERATURE IN ENZYME ACTIVITY Francis Talapian, Stephanie Tantuco, Marionne Tapia, Justin Tiangco, Eileen Visda, *Krisha

Vitto Group 10 2C MT Biochemistry Laboratory ABSTRACT The activity of enzyme is affected by different factors and one of which is the temperature. The aim of the experiment is to determine the effects of the change in temperature on the reaction rates of an enzyme-catalyzed reaction. In this experiment, the invertase was extracted from the yeast and used as the experimental enzyme. Five test tubes with equal amounts of sucrose and invertase when subjected to varying temperatures, and then DNS reagent was added. Each test tube was subjected to 95C to form the ANS, characterized by a red-brown color, then analyzed with the spectrophotometer at 540 nm. The optimum temperature of invertase was found to be 60C. At a certain temperature, the rate of reaction drops drastically which is represented by the bell-shaped curve apparent in the graph. INTRODUCTION Enzymes, proteins that act as catalysts, are the most important type of protein. In the absence of catalysis, most reactions in biological systems would take place far too slowly to provide products at an adequate pace for a metabolizing organism. Catalysts speed up chemical reactions and can go without being used up or changed. Without enzymes, the biochemical reactions that take place will react too slowly to keep up with the metabolic needs and the life functions of organisms. The efficiency of enzymes is affected by a variety of factors, most notably the temperature of the surrounding environment. Temperature, a measure of the intensity of heat, is an important factor in the activity of enzymes. The velocity of an enzymatic reaction is influenced by temperature. This is because substrates collide with active sites frequently in the presence of rapidly moving molecules. In addition, although these molecules do move

rapidly the speed of the reaction drops sharply. In short, thermal agitation causes enzymes to denature or breakdown of protein structures. All enzymes have an optimal temperature at which reaction rates go fastest without denaturing the enzyme. The enzyme that will be used in this experiment is invertase, which is an enzyme that catalyzes the hydrolysis of sucrose. Dinitrosalicylic colorimetric method is a test for the presence of free carbonyl group(C=O), or the so-called reducing sugars. This method involves the oxidation of the aldehyde functional group present in glucose. The reagent used is a yellow dye known as 3,5dinitrosalicylic 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.

Figure 1. Structure dinitrosalicylicacid

of

3,5-

solutions (pH 5), test tubes, pipettes, beakers, volumetric flasks, paraffin film, hot plate, and UV-Vis spectrophotometer. B. Procedure 1. Extraction of Invertase from Yeast 0.25g of Bakers yeast was weighed and distilled water was added until it reached 150 mL. The solution was allowed to stand for 20 minutes at room temperature. Then, the solution was filtered. The supernatant was then used as the enzyme stock solution for the other experiments. 2. Sucrose Assay By Dinitrosalicylic Colorimetric Method A series of test tubes were prepared as seen in the table below (Table 1). 1.5 mL of concentrated HCl was added to each test tube. 1.5 mL of 0.1 M buffer solution at pH 5 was then added to each test tube. Three ml of DNS reagent was added to the mixture. The test tubes were afterward immersed in 95C water bath for 10 minutes to develop the characteristic red-brown color. The test tubes were then cooled and their absorbances were measured at 540 nm. Table 1. Test Tube Preparation for Sucrose Assay Using DNS Colorimetric Method Test Tube mL sucrose mL distilled No. std. solution 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 Figure 2. Test tubes at 95C water bath

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. The objectives of the experiment are to extract invertase that breaks existing bond in sucrose to form glucose and fructose from Bakers yeast and to determine the effects of the change in temperature on the reaction rates of an enzyme-catalyzed reaction. EXPERIMENTAL A. Materials and Compounds Used For the extraction of invertase from yeast, 0.25g Bakers yeast and distilled water were used. For sucrose assay using dinitrosalicylic colorimetric method, sucrose standard solution, distilled water, concentrated HCl, 0.1 M buffer solution (pH 5), DNS reagent, 7 test tubes, and UV-Vis spectrophotometer were used. For the effect of temperature on invertase activity, the following were used: 100mg/L sucrose standard solution, concentrated hydrochloric acid, 0.5M potassium hydroxide, dinitrosalicylic acid (DNS) reagent, 10g/L sucrose solution, 0.1M buffer

3. Determining the Effect of Temperature on Invertase Activity Water baths with temperature of 20C, 30C, 60C, 70C and 90C were prepared. Then, five test tubes with 1.5 mL invertase solution, 1.5 mL of 0.1 M buffer solution at pH 5, and 1.5 mL of sucrose solution were prepared. Afterwards, the test tubes were separately incubated in the water baths prepared earlier for five minutes. Then, 1.5 mL of DNS reagent was added to all test tubes. The test tubes were then immersed in 95C water bath for 10 minutes for the development of the characteristic red-brown color. The solutions were cooled and covered to prevent evaporation. The absorbance was measured at 540nm. Figure 3. Solutions spectrophotometer at UV-Vis

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 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylicacid, 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. To compute for the amount of acid-hydrolyzed sucrose, the equation C1V1 = C2V 2 was used. Using the formula, the following were obtained: At 0.25 ml of sucrose solution

Hydrolyzed Glucose At 0.50 ml of sucrose solution

(100 m g/ m l )(0.25m l ) = ( x)(6 m l) 25 x = =4.17 mg/ml of Acid6 (100 m g/ m l )(0.50 m l ) = ( x)(6 m l) 50 x = =8.33 mg/ml of Acid6 (100 m g/ m l )(0.75m l ) = ( x)(6 m l) 75 x = =12.50 mg/ml of Acid6 (100 m g/ m l )(1.00 m l ) = ( x)(6 m l) 100 x= =16.67 mg/ml of Acid6 (100 m g/ m l )(1.25m l ) = ( x)(6 m l)

Hydrolyzed Glucose At 0.75 ml of sucrose solution

Hydrolyzed Glucose At 1.00 ml of sucrose solution

Hydrolyzed Glucose At 1.25 ml of sucrose solution RESULTS AND DISCUSSION

125 =20.83 mg/ml of Acid6 Hydrolyzed Glucose At 1.50 ml of sucrose solution (100 m g/ m l )(1.50 m l ) = ( x)(6 m l) 150 x= =25.00 mg/ml of Acid6 Hydrolyzed Glucose x=
By measuring the absorbance using UV-Vis Spectrophotometer, the following values were obtained (Table 2) and the standard curve was generated together with the equation through the use of MS Excel (Figure 4). Table 2. Results of the Glucose Assay Using Dinitrosalicyclic Method Test Amount of Absorbance540 Tube AcidNo. Hydrolyzed Glucose (mg/ml) Blank 0 0 1 4.17 0.021 2 8.33 0.030 3 12.50 0.012 4 16.67 0.024 5 20.83 0.030 6 25.00 0.035 Figure 4. Graphical Presentation of Invertase Activity Versus Amount of Acid-Hydrolyzed Glucose

B. Effect of Temperature on Invertase Activity After spectrophotometry, the results were noted down, (Table 3) and graphed (Figure 5). Table 3. Effects Invertase Activity Temperature (C) 20 30 60 70 90 of Temperature in Absorbance540 0.058 0.067 0.074 0.061 0.052

Figure 5. Graphical Presentation of Invertase Activity Versus Temperature.

All enzymes have an optimal temperature at which reaction rates can go faster without denaturing the enzyme. Temperatures at mid range sped up reaction rates of enzymes, but extreme temperatures were not as productive. As shown in the graph shown at Figure 5, there is peak absorbance at 60C. This point is the optimum temperature, where in the enzyme is most active. When the temperature rises, there are more energetic collisions between the enzymes within the reaction. The number of these per minute will also increase, along with the heat of the

molecules. The higher the temperature is, the higher the rate of the enzyme reaction becomes. As it increases, heat is produced. As seen in Figure 5, at a certain temperature, the rate of reaction drops drastically, represented by the bellshaped curve apparent in the graph. This phenomenon can be explained by the nature of the enzymes, which are proteins. All when exposed to enough high temperatures. Once temperatures have reached extreme heat, the enzymes denature, unraveling the protein structure rendering them inactive. Denatured proteins do not react as much as the normal proteins, therefore lessening the reaction rate of the entire system. REFERENCES From Books Boyer, R. F. (2006). Concepts in Biochemistry(3rd ed.). Hoboken, NJ: Wiley. 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 Internet Li, T.S. Effect of temperature on soluble invertase activity, and glucose, fructose and sucrose status of onion bulbs (Allium cepa) in store. (2004) Retrieved January 12, 2013, fromhttp://informahealthcare.com/doi/ab s/10.1080/09637480412331290512. Effects of pH (Introduction to Enzymes).

(n.d.)Retrieved January 12, 2013, from http://www.worthingtonbiochem.com/introBiochem/tempEffects .html. Effect of High Temperature on Sucrose Contentand Sucrose Cleaving Enzyme Activity in RiceGrain During the Filling Stage. (2006) Retrieved January 12, 2013,from http://www.ricescience.org/qikan/manag e/wenzhang/E060309.pdf The Effect Of pH On Invertase Activity. (2003)Retrieved January 12, 2013, from http://www.coursework.info/AS_and_A _Level/Biology/Molecules__Cells/The_ Effect_Of_pH_On_Invertase_Activity_L 107500.html. Wang, N.S. GLUCOSE ASSAYBY DINITROSALICYLIC COLORIMETRIC METHOD Retrieved January 12, 2013, from http://www.eng.umd.edu/~nsw/ench485/ lab4a.htm