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Effects of Change in pH and Temperature on the Reaction Rates of Enzyme-Catalyzed Reaction Marcus Natividad, Barbara Ngo, Lexley Ong

and Jane Jenelle Quilaneta Group 8 2C Pharmacy Biochemistry Laboratory ABSTRACT


In this experiment, the glucose assay using a DNS colorimetric method measured and showed the maximum capacity of invertase activity in increasing concentration of the standard through a graphical representation, the best fit straight line. Factors that affect the invertase is also investigated such as pH and temperature .The results obtained were represented by bellshaped graphs that shows the optimum amount of pH and temperature.

INTRODUCTION This experiment aims to make the students able to determine the effects of change in pH and temperature on the reaction rates of an enzymecatalyzed reaction. Living organisms are composed of intricately systems of chemical reaction. Most of these chemical reactions are catalyzed by enzymes that allow these reactions to proceed at a rate sufficient to sustain life. Enzyme accelerates reactions without being changed themselves [1]. It is protein molecule that is a biological catalyst with three characteristics. First, which is its basic function is to increase the rate of a reaction. 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 [2]. Sucrose, commonly known as table sugar, is a disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,4-glycosidic bond. When this bond is cleaved in a hydrolysis reaction, an equimolar mixture of glucose and fructose is generated. This mixture of monosaccharide is called invert sugar, which is derived from the fact that sucrose rotates plane polarized light to the right i.e., dextrorotatory, +66.5, whereas the hydrolysis products rotates plane polarized light to the left i.e., levorotatory, -20 . Sucrose can be hydrolyzed in the presence of an enzyme called invertase or sucrose which shown in the equation: Sucrose + H2O ---> glucose + fructose The official name for invertase is betafructofuranosidase, which implies that the reaction catalyzed by this enzyme is the hydrolysis of the terminal non-reducing betafructofuranoside residues in betafructofuranosides[3].

Invertase is usually found on plants. It acts as a catalyst for the hydrolysis of sucrose. It is a significant enzyme because glucose is an important product of photosynthesis. Invertase is also used in the confectionery industry where fructose is preferred over sucrose because it is sweeter and does not crystallize easily[4]. EXPERIMENTAL A. Sample Used Sample Used: Glucose, Dinitrosalicyclic reagent B. Procedures 1. Sucrose Assay Using Dinitrosalicyclic Method First, We prepared series of test tubes.All these test tubes were covered with marbles to prevent evaporation of solvent.Then, we added 3 drops (approximately 0.05ml) concentrated HCl to each tube and mixed it. It was incubated at 90C water bath for 5 minutes. Then an addition of 0.15 mL of KOH was done to neutralize the solution. Next, is the addition of 1.5 mL of 0.1 M buffer solution, pH 5 and was mixed.Then , 3 mL of DNS reagent was added. The test tubes were immersed in a 95C water bath for 10 minutes to develop the characteristic red-brown color. After the solution has been cooled, the absorbance at 540nm was measured. Finally, the hydrolizedsucrose standard curve was constructed by plotting A540 against concentration (mg/mL). 2. Effect of pH on Invertase Activity First, we prepared 6 numbered test tubes and a blank test tube. Then, we added 1.4 mL appropriate 0.1 M buffer solution to each test tube. For the blank test tube we just added 3mL of distilled water, for test tube number 1 we added pH 1.00, for test tube number 2 we added pH 3.00, for test tube number 3 we added pH 5.00, for test tube number 4 we added pH 7.00, for test tube number 5 we added pH 9.00 and for test tube number 6 we added pH 11.00. After adding the desired amount of pH to each test tube, 0.10 mL enzyme stock solution was added to each test tube except for the blank test tube.

It was mixed thoroughly and was incubated in 60C water bath for 5 minutes. Next, is the addition 1.50 mL of sucrose solution and was incubated reaction mixture in 60C water bath for 5 minutes. Then, 3 mL of Dinitrosalicyclic reagent was added. The test tubes were immersed in 5C water bath for 10 minutes to develop the characteristic red-brown color. Then the absorbance at 540 nm was measured. Finally, the amount of sucrose hydrolyzed was determined using hydrolyzed-sucrose standard curve that was constructed in the Dinitrosalicyclic colorimetric method. 3. Effect of Temperature on Invertase Activity To test the effect of temperature on Invertase activity, our group placed 20, 30, 50, 60, 70 and 90C water baths and prepared 6 test tubes with each test tube containing 1.5 mL sucrose solution. The test tubes were incubated separately for 5 minutes in each water bath. In another test tube, 0.80 mL enzyme stock solution was mixed with 19.20 mL 0.1M buffer solution,pH 5. Then, we added 3 mL of diluted enzyme solution to all tubes. It was incubated for another 5 minutes. Remember not to remove the test tubes from their respective water baths. Then, 3 mL of DNS reagent was added. The test tubes were immersed in a 95C water bath for 10 minutes to develop characteristic re-brown color and then it was cooled down. Remember that all the test tubes are covered with marble to prevent evaporation of solvent. Meanwhile, another member prepared a bank solution by repeating all the steps but instead of adding enzyme stock solution, denatured enzyme is used. Then, the absorbance at 540nm is measured. The amount of sucrose hydrolyzed was determined by using sucrose standard curve constructed in the dinitrosalicylic colometric method. RESULTS AND DISCUSSION Glucose, and other reducing sugars,reacts with DNS by oxidation reaction producing a colored compound that shows a maximal molar extinction at 530nm.Glucose is reduced into gluconic acid,same as to dinitrosalicylic acid to 3-amino,5-nitro saliccycli acid,giving the characterisctic red-brown color. Oxidation is a reversible chemical reaction in which one of the reactions is oxidation and the reverse reaction is reduction. Since the absorbance at 540 nm is linearly dependent on the concentration or mass of glucose the reaction can be used for quantification of reducing sugar In table 1, We were able to measure the amount of light transmitted through a sample at a given wavelength. The absorbance of blank test tube was subtracted to the

absorbance of the numbered test tubes. This was made to measure only the absorbance of the invertase, without the additional reagents.
Test tube no Amount of Acid hydrolyzed glucose (mg/ml)

Absorbance

blank 1 2 3 4 5 6 Table 1.Glucose Method

0 0.5 0.83 1.33 1.67 2.17 2.50 Assay Using

2.35 2.10-2.35 =-0.25 2.96-2.35 =0.61 3.04-2.35 =0.69 3.07-2.35 =0.72 3.10-2.35 =0.78 3.11-2.35 =0.76 Dinitrosalicyclic

The graph below is the glucose calibration curve. It was shows the presence of glucose in the solution and the rate of formation of glucose. It is illustrated that the absorbance is directly proportioned to the amount of hydrolyzed glucose which means that the higher the absorbance the higher amount of acid hydrolyzed glucose is present.

1 0.8 Absorbance 0.6 0.4 0.2 0 0

Glucose Assay

Glucose mg/ ml Figure 2. Linear Graph of Glucose Assay Using Dinitrosalicylic Method The Invertase can be affected by the changes in pH. An Extremely high or low pH values generally result in complete loss of activity for most enzymes.

pH

Amount of Acid hydrolyzed sucrose (mg/ml) 0.5

Absorbance 30 0.1

=1.126 1.459-0.213 =1.246 50 0.12 1.52-0.213 =1.367 60 0.32 2.88-0.213 =2.667 70 0.18 1.799-0.213 =1.586 90 0.17 1.762-0.213 =1.549 Table 3. Effect of Temperature on Invertase activity Optimum Temperature was obtained at 60C . It infers that the activation energy is spontaneous that the transition state was inferred to the highest value of absorbance in the graph. The highest peak of the curve represents the highest concentration which was determined by the Absorbance. Temperature above 60C shows that the invertase reached denaturation. Effect of temperature 0.35 0.3 mg/ml/min 0.25 0.2

blank 3 4 5 7 8 11 Table

0.223 A

1.652-0.233 =1.419 A 0.34 1.652-0.233 =0.032 A 0.28 1.580-0.233 =1.347 A 0.36 1.660-2.33 =1.427 A 0.26 1.601-0.233 =1.368 0.02 1.459-0.233 =1.226 2. Effect of pH on Invertase Activity

In figure 3, A bell shaped graph was formed. The most favorable pH value, which is the point where the enzyme is most active, is known as the optimum pH is at pH 6. It shows that at pH 7 and above it the concentration of invertase will slope down indicating denaturation of enzyme. It indicates that invertase reactivity is controlled and occurs to alimited extent. Effects of pH 1.4 1.2 mg/mL/min 1 0.8 0.6 0.4 0.2 0 0 5 pH 10 15

0.15 0.1

0.05 0 0 50 Temperature 100

Figure 4. Graph of Temperature on Invertase Activity REFERENCES [1]Crisostomo A. et.al.(2010).Laboratory Manual in General Biochemistry.Quezon City: C&E Publishing Inc.do.edu/hndbksupport/ochemlabtech.html 2003 [2]http://www.elmhurst.edu/~chm/vchembook/5 70enzymes.html [3]http://www.eng.umd.edu/~nsw/ench485/lab1 4.htm [4]Campbell N.A.;Reece J.B..(2002).Biology,6thedition.San Francisco,USA: Benjamin Cummings.

Figure 3. Graph of the effect of pH on Invertase Activity The temperature affects the invertase activity. Too cool will not allow the enzyme to react, Too hot will denature the protein.

Temperature

20

Amount of Acid hydrolyzed sucrose (mg/ml) 0.05

Absorbance

1.339-0.213