Biochemistry Laboratory PH-BIOCHEM (2012 2013) 2B-Ph Group 7 Experiment 3

INVESTIGATING THE FACTORS THAT AFFECT ENZYME ACTIVITY AND THEIR CORRESPONDING EFFECTS Nano, Lizette A., Olivete, Lesli Linka Mel L., Ong, Feliz Jem V., Ong, Ralph Timothy S., Ortega, Aira Marie A.* Date submitted: January 7, 2012

Abstract
Enzymes are biological catalysts; they can help in speeding up the rate of reaction or rather alter it. Enzymes are affected by two factors: pH and temperature. This experiment aims to find out and investigate the effects of these two factors in the enzyme activity. Along the way, the group will find out what values of these factors at which the enzyme activity will be at its most favourable condition. These values are called optimum pH and optimum temperature. Extreme values of these two factors lead to the alteration in the structure and activity of enzymes. The paper will be focusing more of one of the factors, the pH. Invertase was subjected to different PH of buffer solution and was observed under 540 nm absorbance using spectrophotometer. Dinitrosalicyclic acid (DNS) Assay method is utilized to determine the amount of acid hydrolyzed sucrose. The samples in the assay method were also observed under 540 nm absorbance using the spectrophotometer.

Introduction Enzymes are proteins that participate in cellular metabolic processes with the ability to enhance the rate of reaction between molecules. They consist of various types of proteins that work to drive the chemical reaction required for a specific action or nutrient. Enzymes are classified according to the reactions they hydrolyse. There are six classes of enzymes namely: a.) Oxidoreductases b.) Transferases c.) Hydrolases d.) lyases e.) Isomerases f.) Ligases Enzymes are affected by pH and temperature. Extreme high or low pH values generally result in complete loss of activity for most enzymes. In addition,

pH plays an important role in the stability of enzymes.

Figure 1.0

Optimum pH value is defined as the most favourable pH value-point wherein the enzyme is most active and has its highest reaction rate. The optimum pH value varies from one enzyme to another. The bell shaped curve in table Figure 1.0 illustrates the optimum pH.

Low pH describes the slow reaction rate of enzymes. High pH, on the other hand, demonstrates the almost same result but this time it shows the loss of enzymatic activity. Another factor that affects enzyme activity is temperature. Enzymes also have their preferred temperature in which their reaction rates are in their maximum; without having them being denatured or broken down. This preferred temperature is called optimum temperature. The optimum temperature of enzyme is around 37 . Enzymes are said to be denatured at around 40 . DNS reagent, or Dinitrosalicylic Acid (3,5-Dinitrosalicylic Acid) is a yellow reagent generally used for determination of reducing sugars. It is an aromatic compound that reacts with reducing sugars and other reducing molecules to produce 3-Amino-5-nitrosalicylic acid (which is reddish brown in color). DNS usually indicates the presence of free carbonyl groups, the so-called reducing sugars. Sucrose is an organic compound commonly referred to as table sugar or sometimes, saccharose. It is a disaccharide composed of monosaccharides glucose and fructose. Glucose, which is a reducing sugar, will react with DNS due to its free carbonyl group present in its structure. Solutions of high sucrose concentration will show darker, reddish brown color. METHODOLOGY A. Sucrose Assay Using Dinitrosalicylic Colorimetric Method Prepare a series of test tubes as follows:

Table 1.0- Test tube preparation for the Sucrose Assay

Tube No.

Volume of Sucrose Standard Solution

Volume of Distilled Water

Blank 0 1.5 1 0.25 1.25 2 0.50 1.00 3 0.75 0.75 4 1.00 0.5 5 1.25 0.25 6 1.50 0 *Cover all tubes with marbles to prevent evaporation of solvent. Add three (3) drops (0.05 mL) concentrated Hydrochloric Acid to each tube. Mix well. Incubate at 90 water bath for five minutes. Add 0.15 mL of 0.5 M Potassium Hydroxide to neutralize the solution. Add 2.80 mL of 0.1 M buffer solution, pH. Mix well. Add 3 mL of Dintrosalicylic Acid (DNS Reagent). Immerse the test tubes in 95 water bath for ten minutes to develop the characteristic red-brown color. After cooling, measure the absorbance at 540 nm. Construct the 2ydrolysed-sucrose standard curve by plotting A540 against concentration (mg/mL). B. Extraction of Invertase from Yeast Weigh 0.25 g Bakers Yeast. Dissolve in distilled water to make a 250-mL solution. Allow the solution to stand for twenty minutes at room temperature. Collect the supernatant if sedimentation occurs. The supernatant serves as the enzyme stock solution that will be used for succeeding experiments.

C. Preparation of Denatured Invertase Stock solution Incubate 100 mL enzyme stock solution in a boiling water bath for ten minutes. Allow the solution to cool. Collect only the supernatant if frothing occurs. This serves as the denatured enzyme stock solution that will be used for the succeeding experiments. D. Effect of pH on Invertase Activity Prepare six numbered test tubes. Add 2.90 mL appropriate to 0.1 M buffer solution as described below. Label the test tubes accordingly.
Table 2.0- Test tube preparation for the Effect of pH on Invertase Activity

E. Effect of Temperature on Invertase Activity Set up 20, 30, 50, 60, 70 and 90 water baths. Prepare six test tubes with each test tube containing 1.5 mL sucrose solution. Incubate the test tubes separately for five minutes in each water bath. In another test tube mix 0.80 mL enzyme stock solution with 19.20 mL of 0.1 M buffer solution, pH 5. Add 3 mL of dilute enzyme solution to all tubes. Incubate for another five minutes. *Do not remove the test tubes from their respective water baths. Add 3 mL of DNS Reagent. Immerse the test tubes in 95 water bath for ten minutes to develop the characteristic red brown color. Allow the solutions to cool. *Cover all tubes with marbles to prevent evaporation of solvent Prepare blank solutions by following steps 2-5. Add denatured enzyme instead of enzyme stock solution. Measure the absorbance at 540 nm. Determine the amount if sucrose 3ydrolysed using the sucrose standard curve constructed in the dinitrosalicylic colorimetric method. RESULTS AND DISCUSSION A. Sucrose Assay using Dinitrosalicylic Colorimetric Method Dinitrosalicylic Acid (DNS reagent) was used in this particular part of the experiment, Sucrose assay. As mentioned before, DNS is a yellowcolored reagent that is used for determining reducing sugars in a compound. It is an aromatic compound that reacts with reducing sugars (those

Tube No. 1 2 3 4 5 6

pH of Buffer Solution 1 3 5 7 9 11

Add 0.10 mL enzyme stock solution to each test tube. Mix thoroughly. Incubate all tubes in 60 water bath for five minutes. Add 1.50 mL of sucrose solution and incubate reaction mixture in 60 water bath for five minutes. Add 3 mL of Dinitrosalicylic Acid (DNS Reagent). Immerse the test tubes in 95 water bath for ten minutes to develop the reddish brown color. Allow the solutions to cool. Prepare the blank solutions by following steps 1-4. Add denatured enzyme instead of enzyme stock solution. Measure the absorbance at 540 nm. Determine the amount of sucrose 3ydrolysed using the 3ydrolysed-sucrose standard curve constructed in the dinitrosalicylic colorimetric method.

with free carbonyl group) to form 3Amino-5-nitrosalicylic acid. A positive result yields a red-brown color of the solution. In the experiment, the group prepared the six test tubes as stated by the procedure. DNS was added to the mixture of potassium hydroxide, concentrated hydrochloric acid, buffer solution and sucrose solution. Concentrated hydrochloric acid was added to the sucrose solution since sucrose will not undergo a reaction with DNS. The former must be first broken down into smaller molecules. This can be done by adding concentrated Hydrochloric acid and boiling the solution. Then potassium hydroxide and the buffer solution are added to adjust the pH, making the solution basic. Under this condition, simple sugars are good reducing agents. As a result, the group yielded the expected red brown color of the prepared solutions. This only proves that indeed 3-Amino-5-nitrosalicylic acid was formed due to the presence of a reducing sugar. Glucose was the reducing sugar present in the mixture prepared; since it is one of the major components of sucrose.
Figure 2.0

The theoretical amount of the acidhydrolyzed sucrose was computed using the equation C1V1=C2V2. Below are the obtained quantities.

Test tube 1 0.1mg/mL(0.25mL)=1.5mL x X=0.017mg/mL Test tube 2 0.1mg/mL(0.50mL)=1.5mL x X=0.033mg/mL Test tube 3 0.1mg/mL(0.75 mL)=1.5mL x X=0.050mg/mL Test tube 4 0.1mg/mL(1.00 mL)=1.5mL x X=0.067mg/mL Test tube 5 0.1mg/mL(1.25mL)=1.5mL x X=0.250mg/mL Test tube 6 0.1mg/mL(1.50mL)=1.5mL x X=0.100mg/mL
Table 3.0- The amount of acid-hydrolyzed sucrose (theoretical) and the measurement of absorbance at 540 nm by the spectrophotometer. Amount of acidAbsorbance at hydrolyzed sucrose 540nm (theoretical)

The absorbance of the acid-hydrolyzed sucrose in each test tube was determined through the use of the UVvis spectrophotometer.

0 0.017 0.033 0.050 0.067 0.250 0.100

0 0.212 0.158 0.196 0.169 0.158 0.128

Figure 3.0

the electronic state of these amino acids. When this happens, an ionic bond can either be formed or broken. Thus results a distorted protein. A catalytic center in the enzyme can be deactivated by extreme pH changes, causing the enzyme activity to slow down or lose function. The experiment aimed to find out at which pH the enzyme activity will be at its best and which it will be in decline.

Theoretically, the line represents the expected plot of the graph in the experiment. A linear trend should be seen in the graph to ensure that the results obtained are correct. But the graph plotted has shown otherwise. There must have been certain errors that were made during the experiment that affected the results gathered. The following are suspected errors: a. inaccurate measurement of the samples b. contamination of some samples c. failure to completely comply with the procedures B. Effect of pH on Invertase/Enzyme Activity pH plays an relevant role in enzyme activity. There is what they call the optimum pH- the most favourable pH for an enzyme. It is where the enzyme is most active and has the highest reaction rate. Values below the optimum pH will mean a slow reaction rate for enzyme. On the other hand, those of extreme high pH value will mean, generally, loss of enzyme activity. This denatures enzymes. Enzymes generally contain amino acids that are sensitive to pH in structural and catalytic regions. Changing the pH alters

A bell shaped graph is expected to result from the experiment. This ensures the accuracy of the gathered data since it shows the optimum pH (peak of enzyme activity), lower and higher pH values than the optimum pH (Lower pHwhich describes the slow reaction rate for enzymes before achieving optimum pH; higher pH/extreme high pH- which describes the decline of the enzyme activity where the enzyme denatures.). From the results obtained by the group below, it shows that the optimum pH for enzymes is 7. Enzymes perform well at pH 3-7 while beyond those values meant denaturation and loss of enzyme activity. The following results were obtained from the said experiment.
pH Absorbance at 540 nm

1 3 5 7 9 11

0.018 0.037 0.054 0.082 0.063 0.047

Figure 4.0

REFERENCES: 1. Philipps, Theresa; Enzymes http://biotech.about.com/od/glossary/g/Enzyme.htm 2. http://www.angelfire.com/de/nestsite/modbio05.html Manual of Clinical enzyme Measurement, 1972; Introduction to Enzymes http://www.worthingtonbiochem.com/introBiochem/Enzymes.pdf 4. Biology:How Temperature Affects Enzymes http://www.woisd.net/moodle/mod/resource/view.php? id=44 http://www.scribd.com/doc/58234422/Formal-ReportExperiment-3-Enzymes 5. Glucose Assay by DNS Method http://faculty.ksu.edu.sa/aabulhamd/Documents/II%20 lab/GLUCOSE%20ASSAY%20(353).pdf 3.