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ENZYMES: BIOLOGICAL CATALYSTS Alabastro, Reena Avemiel, Albeza, Keeshan Gytha, Arcinas, Ann Iree, Baello, Alexa Jeatrize,

*Chamzelle Carlos, Chia, Anna Patricia Group 1- 2GPH 2013 Faculty of Pharmacy University of Sto. Tomas

ABSTRACT: Enzyme is any of several complex proteins that are produced by cells and act as catalysts in specific biochemical reactions. [1] Thousands of different enzymes can be found within a cell, and each has a unique three-dimensional structure that dictates its function. The rate of reaction of enzyme varied as the enzyme changed in shape and structure which is caused by the factors that affect the rate of reaction of the enzymatic activity. In this experiment, Invertase was extracted from yeast and the collected supernatant serves as the stock solution that was used in the preparation of denatured invertase stock solution. Sucrose assay was performed using Dinitrosalicylic acid Colorimetric Method which is a technique used to monitor or determine enzyme activity; then the effect of PH and temperature on invertase activity was determined. At the end of the experiment, the graph of the Absorbance and the amount of Acid Hydrolyzed Sucrose using Sucrose Assay were determined; with the aid of the slope and intercept from the graph of Sucrose Assay, the Amount of Acid Hydrolyzed was determined versus pH and temperature. Hence, it was identified that exactly high/ low pH values generally result in a complete loss of activity for most enzyme. In terms of temperature, too cool will not allow the enzyme to react while too hot will denaturate the enzyme. INTRODUCTION [2] An enzyme is a protein molecule that is a biological catalyst with three characteristics. First, the basic function of an enzyme is to increase the rate of a reaction. Most cellular reactions occur about a million times faster than they would in the absence of an enzyme. Second, most enzymes act specifically with only one reactant (called a substrate) to produce products. The third and most remarkable characteristic is that enzymes are regulated from a state of low activity to high activity and vice versa. Enzymatic activity is affected by different factors such as pH, temperature, enzyme concentration (when [S] is constant), substrate concentration (when [E] is constant), enzyme inhibitors/activators/ cofactor/coenzymes.
Enzymes are sensitive by changes in pH and temperature. [3] The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH. This is graphically illustrated in Figure 1.

Figure 1. Optimum pH [4] Several factors are influenced directly by the pH in which the reaction takes place these are: the binding of substrate to the enzyme, the ionization states of the amino acid residues involved in the catalytic activity of the enzyme, the

ionization of the substrate variation in the protein structure at extreme pH. Dependence on pH of enzyme activity is a consequence of acid-base behavior or changing degree of ionization of groups in the enzyme, in the substrate, or in both. Every enzyme has a temperature range of optimum activity. Temperature can affect an enzyme in two ways: one is a direct influence on the reaction rate constant, and the other is in thermal denturization of the enzyme at elevated temperatures. Rate of reaction increases with temperature; within the temperature range in which the enzyme is stable and retains its full activity. When temperature range is beyond 10-50 at optimum, enzymes are denatured followed by a decrease in enzymatic activity. [5] Dinitrosalicylic acid (DNS) Assay is a method used to determine enzyme activity. In this assay the DNS (3,5-dinitrosalicylic acid, IUPAC name: 2-hydroxy-3,5-dinitrobenzoic acid) reacts with reducing sugars to form 3-amino-5-nitrosalicylic acid (ANS) which absorbs light strongly at 540 nm. The rate of reaction is monitored (colorimetrically) by measuring the amount of reaction products that reacts with DNS reagent. The objective of the study is to (1) extract invertase from Bakers yeast; (2) to determine the effects of changes in PH and temperature on reaction rates of an enzyme-catalyzed reaction. METHODOLOGY A. Sucrose Assay Using Dinitrosalicylic Colorimetric Method Series of test tubes were prepared as seen in Table 1. Three drops (0.05 mL) of concentrated HCL was added to each test tube. The tubes were mixed well and incubated at 90C water bath for 5 minutes. 0.15 mL 0.5 M KOH was added to neutralize the solution. A 2.80 mL 0.1 M buffer solution, pH 5 was added and mixed well. 3 mL of DNS reagent was added. The test tubes were immersed in 95C water bath for 10 minutes to develop the characteristic red-brown color. The absorbance was then measured at 540 nm after the cooling test tubes. Table 1 Test tube preparation for Sucrose Assay Using Dinitrosalicylic Colorimetric method
Tube no. mL sucrose standard solution mL distilled water Blank 1 2 3 4 5 6

0.25

0.50

0.75

1.00

1.25

1.50

1.50

1.25

1.00

0.75

0.50

0.25

B. Effect of pH on Invertase Activity Six numbered test tubes were prepared. 2.90 mL appropriate 0.1M buffer solution was added in each test tube with different pH as described in Table 2. 1.50 mL enzyme stock solution was added to each test tube. The tubes were mixed and then incubated in 60 water bath for 5 minutes. 1.50 mL of sucrose solution was asses and the reaction mixtures were incubated in 60 water bath for 5 minutes. A three mL of DNS reagent was then added. The test tubes were immersed in 95 water bath for 10 minutes to develop the characteristic red-brown color. Then the solutions were cooled. Blank solutions were prepared by following steps 1- 4. Denatured enzyme was added instead of enzyme stock solution. The absorbance was then measured at 540 nm. Table 2 Test tube preparation for effect of pH on Invertase Activity Tube 1 2 3 4 5 6 no. pH of 0.1 0.3 0.5 1.7 1.9 1.11

buffer solution C. Effect of Temperature on Invertase Activity 20, 30, 50, 60, 70, and 90 water baths were set-up. Six test tubes were prepared with each test tube containing 1.5 mL sucrose solution. The test tubes were incubated separately for 5 minutes in each water bath. 0.80 mL enzyme stock solution with 19.20 mL 0.1 M buffer solution, pH 5 was then mixed in another test tube. Three mL of dilute enzyme solution was added to all tubes and were incubated again for 5 minutes. A three mL of DNS reagent was then added. The test tubes were immersed in 95 water bath for 10 minutes to develop the characteristic red-brown color. Blank solutions were prepared by following steps 2- 5. Denatured enzyme was added instead of enzyme stock solution. The absorbance was then measured at 540 nm. RESULTS AND DISCUSSION A. Sucrose Assay Using Dinitrosalicylic Colorimetric Method As the dinitrosalicylic acid, a yellow reagent was added in tubes with the existence of heat, a red-brown color was observed in the mixture. This is due to the reaction of DNS with reducing sugars to form 3-amino-5-nitrosalicylic acid (ANS) which contributed to the redbrown coloration. However, DNS does not react with sucrose (non-reducing sugar). The absorbance of each hydrolyzed-sucrose is determined by UV-vis spectrophotometer. Using the formula C1V1=C2V2, the amount of acid-hydrolyzed sucrose were computed as follows: Blank test tube: C2 = Test tube 1: C2 =
( ( )( )

)(

Test tube 2: C2 =
( )( )

Test tube 3: C2 =
( )( )

Test tube 4: C2 =
( )( )

Test tube 5: C2 =
( )( )

Test tube 6:

C2 =

)(

Table 3. x and y values for amount of Acid-Hydrolyzed Sucrose (mg/mL) and Absorbance540 Amount of Acid-Hydrolyzed Absorbance540 Test Tube No. Sucrose (mg/mL) Blank 0 0 1 0.00556 0.0151 2 0.01111 0.013 3 0.01667 -0.006 4 0.02222 0.170 5 0.02778 0.183 6 0.02556 0.226
0.25 0.2 0.15 Absorbance540 0.1 0.05 0 0 -0.05 -0.1 Amount of Acid-Hydrolyzed Sucrose (mg/mL) 0.005 0.01 0.015 0.02 0.025 0.03 y = 8.2436x - 0.0424 R = 0.7167

Figure 3. Absorbance vs. Amount of Acid-Hydrolyzed sucrose The line represents the best fit line, [6] a line on a graph showing the general direction that a group of points seem to be heading. The slope-intercept form was computed to be y=8.2436x0.0424. Factors such as inactivity of sucrose if it is not freshly prepared, DNS might not be reactive or the spectrophotometer is defective or not sensitive enough may be the cause of non-linear trend identified. B. Effect of pH on Invertase Activity y= absorbance b= intercept m=slope

The intercept was -0.424 , while the slope was 8.2436. These will be used to compute the amount of acid-hydrolyzed sucrose. Table 4. Amount of Acid Hydrolyzed Sucrose and Absorbance (at 540 nm) at certain pH Amount of Acid Hydrolyzed pH Absorbance540 Sucrose (mg/mL) Blank 0 0 2 0.0093 0.0345 3 0.0247 0.161 5 0.0229 0.1465 7 0.0284 0.1915 7.5 0.0390 0.2785 8 0.0078 0.022 9 0.0099 0.0395 11 0.0079 0.0225 At pH 2: ( ( ) )

At pH 3:

At pH 5:

( ( ( ( ( (

) ) ) ) ) )

At pH 7:

At pH 7.5:

At pH 8:

At pH 9:

At pH 11:

0.3 Amount of Acid Hydrolyzed Sucrose 0.25 0.2 0.15 0.1 0.05 0 0 2 4 6 pH 8 10 12

Figure 4. Amount of Acid Hydrolyzed vs pH [7] 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 enzyme. The result of experimental graph is close from the ideal graph which is a bell shaped graph. This curve describes the stability of enzymes during the alteration of pH. A low pH results to slow reaction rate but too high pH shows the same result due to enzymatic loss. The peak of the graph determines the best pH suitable for the enzyme that it reaches the maximum reaction rate. In this graph the best pH is 0.28.

B. Effect of Temperature on Invertase Activity

y= absorbance b= intercept m=slope The intercept was -0.424 , while the slope was 8.2436. These will be used to compute the amount of acid-hydrolyzed sucrose. Table 5. Amount of Acid Hydrolyzed Sucrose and Absorbance (at 540 nm) at certain temperature. Amount of Acid Hydrolyzed Temperature Absorbance540 Sucrose (mg/mL) Blank 0 0 20 0.00799 0.0235 30 0.0223 0.1415

50 50 60 60 70 90 At temperature 20:

0.0144 0.0338 0.0504 0.0227 0.0051 0.0051 ( )

0.0765 0.2365 0.373 0.145 0 0

At temperature 30:

( ( ( ( ( ( )

) ) ) ) )

At temperature 50:

At temperature 50:

At temperature 60:

At temperature 60:

At temperature 70:

At temperature 90:

Amount of Acid Hydrolyzed Sucrose

0.06 0.05 0.04 0.03 0.02 0.01 0 0 20 40 60 80 100 Temperature

Figure 5. Amount of Acid Hydrolyzed vs.Temperature


Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. [8] A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. As shown in Figure 5, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most enzymes rapidly become denatured at temperatures above 40C, most enzyme determinations are carried out somewhat below that temperature.

REFERENCES [1] Crisostomo, A.C., Daya, M.L., de Guia, R. M., Farrow, F.L., Gabona, M.G., Liu, M.I., Pena, G.T., Pena, L.L., Santiago, L.A., Santiago, M.R., Sarile, A.S., Torres, P.C., Vargas, A.G., Ysrael, M.C. (2010). Laboratory Manual in General Biochemistry. Phi: C&E Publishing, Inc. Page 41. (Retrieved last January 14,2014) [2] Ophardt, C.E.(2003). Virtual Chembook. Elmhurst College. (Retrieved last January 14,2014) [3] Pfeiffer, J.: Enzymes, the Physics and Chemistry of Life, pg 171-173, Simon and Schuster, NY (1954). (Retrieved last January 14,2014) [4] Voet, Voet and Pratt, Fundamentals of Chemistry, John Wiley & Sons, Inc. 1999 Blanch and Clark, Biochemical Engineering, Marcel Dekker, Inc. 1997. (Retrieved last January 14,2014)

[5]

Chul-, W.P., Zipp, E. (1999). http://www.rpi.edu/dept/chem-eng/BiotechEnviron/Projects00/temph/enzyme.html The effects of Temperature and pH on Enzyme Activity. (Retrieved last January 14,2014) [6] Copyright 2011 MathsIsFun.com. http://www.mathsisfun.com/definitions/line-of-bestfit.html. (Retrieved last January 14,2014) [7] Martinek, R.: Practical Clinical Enzymology: J. Am. Med. Tech., 31, 162 (1969).
(Retrieved last January 14,2014)

http://www.chem.fsu.edu/chemlab/bch4053l /enzymes/activity/index.html

[3] Bellis, M. http://inventors.about.com/library/inventors/ blaspirin.htm, The History of Aspirin, 15 July 2009. (Retrieved last September 28, 2013) [4] Whitten, K. W., R. E. Davis, M. L. Peck, G. G. Stanley (2007). Chemistry. 8th ed. USA: Thomson Brooks/Cole, p. 947. (Retrieved last September 28, 2013)

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