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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
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.
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.
The supernatant serves as the enzyme stock solution that will be used for succeeding experiments. Add three (3) drops (0. pH.5 M Potassium Hydroxide to neutralize the solution.00 0. reddish brown color. Collect the supernatant if sedimentation occurs.25 g Baker‟s Yeast. demonstrates the almost same result but this time it shows the loss of enzymatic activity.80 mL of 0.25 1. This „preferred‟ temperature is called optimum temperature.75 0.0. Mix well. saccharose.1 M buffer solution. or Dinitrosalicylic Acid (3. It is a disaccharide composed of monosaccharides glucose and fructose.05 mL) concentrated Hydrochloric Acid to each tube.5 1 0. measure the absorbance at 540 nm.15 mL of 0. Incubate at 90 water bath for five minutes. After cooling. Glucose. DNS usually indicates the presence of free carbonyl groups. will react with DNS due to its free carbonyl group present in its structure.00 3 0.Test tube preparation for the Sucrose Assay Tube No. Sucrose Assay Using Dinitrosalicylic Colorimetric Method Prepare a series of test tubes as follows: Table 1. Enzymes are said to be denatured at around 40 . The optimum temperature of enzyme is around 37 . Sucrose is an organic compound commonly referred to as table sugar or sometimes. Construct the 2ydrolysed-sucrose standard curve by plotting A540 against concentration (mg/mL). without having them being denatured or broken down. 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). Add 2. on the other hand. .25 6 1. Allow the solution to stand for twenty minutes at room temperature.5 5 1.75 4 1. Solutions of high sucrose concentration will show darker.Low pH describes the slow reaction rate of enzymes. Another factor that affects enzyme activity is temperature.50 0 *Cover all tubes with marbles to prevent evaporation of solvent. Extraction of Invertase from Yeast Weigh 0.5-Dinitrosalicylic Acid) is a yellow reagent generally used for determination of reducing sugars. Dissolve in distilled water to make a 250-mL solution.25 2 0. Add 0. Enzymes also have their „preferred‟ temperature in which their reaction rates are in their maximum. B. Mix well.25 0. the so-called reducing sugars.50 1. High pH. DNS reagent. METHODOLOGY A. 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. which is a reducing sugar. Volume of Sucrose Standard Solution Volume of Distilled Water Blank 0 1.
50.90 mL appropriate to 0. Prepare the blank solutions by following steps 1-4. Immerse the test tubes in 95 water bath for ten minutes to develop the characteristic red brown color. 30.20 mL of 0. Sucrose assay. Sucrose Assay using Dinitrosalicylic Colorimetric Method Dinitrosalicylic Acid (DNS reagent) was used in this particular part of the experiment. Effect of pH on Invertase Activity Prepare six numbered test tubes.5 mL sucrose solution. DNS is a yellowcolored reagent that is used for determining reducing sugars in a compound. Label the test tubes accordingly. Incubate all tubes in 60 water bath for five minutes.1 M buffer solution as described below. Allow the solutions to cool. Add denatured enzyme instead of enzyme stock solution. Determine the amount if sucrose 3ydrolysed using the sucrose standard curve constructed in the dinitrosalicylic colorimetric method. Table 2. Immerse the test tubes in 95 water bath for ten minutes to develop the reddish brown color. Measure the absorbance at 540 nm. Add denatured enzyme instead of enzyme stock solution. This serves as the denatured enzyme stock solution that will be used for the succeeding experiments. Allow the solutions to cool. . Add 3 mL of DNS Reagent. D. Add 2. 60. Effect of Temperature on Invertase Activity Set up 20.0. 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. *Do not remove the test tubes from their respective water baths. Add 3 mL of Dinitrosalicylic Acid (DNS Reagent).80 mL enzyme stock solution with 19. Preparation of Denatured Invertase Stock solution Incubate 100 mL enzyme stock solution in a boiling water bath for ten minutes. Prepare six test tubes with each test tube containing 1.Test tube preparation for the Effect of pH on Invertase Activity E. Incubate the test tubes separately for five minutes in each water bath. *Cover all tubes with marbles to prevent evaporation of solvent Prepare blank solutions by following steps 2-5. pH 5.10 mL enzyme stock solution to each test tube. Collect only the supernatant if frothing occurs. Mix thoroughly. As mentioned before. Incubate for another five minutes. Add 3 mL of dilute enzyme solution to all tubes. In another test tube mix 0. Determine the amount of sucrose 3ydrolysed using the 3ydrolysed-sucrose standard curve constructed in the dinitrosalicylic colorimetric method. Measure the absorbance at 540 nm. RESULTS AND DISCUSSION A.50 mL of sucrose solution and incubate reaction mixture in 60 water bath for five minutes. 70 and 90 water baths.1 M buffer solution.C. Allow the solution to cool. Add 1.
00 mL)=1.100mg/mL Table 3.1mg/mL(1.1mg/mL(1.033 0.0.5mL x X=0.1mg/mL(1. This can be done by adding concentrated Hydrochloric acid and boiling the solution. DNS was added to the mixture of potassium hydroxide.067mg/mL Test tube 5 0. Then potassium hydroxide and the buffer solution are added to adjust the pH.196 0.250mg/mL Test tube 6 0.158 0.25mL)=1.0 The theoretical amount of the acidhydrolyzed sucrose was computed using the equation C1V1=C2V2.50mL)=1.5mL x X=0.100 0 0.050 0.1mg/mL(0.250 0.with free carbonyl group) to form 3Amino-5-nitrosalicylic acid. As a result.169 0. This only proves that indeed 3-Amino-5-nitrosalicylic acid was formed due to the presence of a reducing sugar. The former must be first broken down into smaller molecules. Test tube 1 0.25mL)=1.5mL x X=0.50mL)=1. the group prepared the six test tubes as stated by the procedure.212 0.5mL x X=0. In the experiment. the group yielded the expected red brown color of the prepared solutions. Figure 2.017 0.050mg/mL Test tube 4 0. since it is one of the major components of sucrose. concentrated hydrochloric acid.1mg/mL(0.5mL x X=0.128 . A positive result yields a red-brown color of the solution. 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.The amount of acid-hydrolyzed sucrose (theoretical) and the measurement of absorbance at 540 nm by the spectrophotometer. Glucose was the reducing sugar present in the mixture prepared. simple sugars are good reducing agents.067 0. 0 0.5mL x X=0.033mg/mL Test tube 3 0. buffer solution and sucrose solution.1mg/mL(0. making the solution basic.158 0. Under this condition.75 mL)=1. Below are the obtained quantities.017mg/mL Test tube 2 0. Concentrated hydrochloric acid was added to the sucrose solution since sucrose will not undergo a reaction with DNS.
037 0. But the graph plotted has shown otherwise.063 0. The following results were obtained from the said experiment. A catalytic center in the enzyme can be deactivated by extreme pH changes. loss of enzyme activity.082 0. It is where the enzyme is most active and has the highest reaction rate. it shows that the optimum pH for enzymes is 7. The following are suspected errors: a.). higher pH/extreme high pH. This ensures the accuracy of the gathered data since it shows the optimum pH (peak of enzyme activity).018 0.047 .0 the electronic state of these amino acids. Changing the pH alters A bell shaped graph is expected to result from the experiment. A linear trend should be seen in the graph to ensure that the results obtained are correct. The experiment aimed to find out at which pH the enzyme activity will be at its best and which it will be in decline.which describes the decline of the enzyme activity where the enzyme denatures. causing the enzyme activity to slow down or lose function. Theoretically. Enzymes generally contain amino acids that are sensitive to pH in structural and catalytic regions. contamination of some samples c. There must have been certain errors that were made during the experiment that affected the results gathered. On the other hand. There is what they call the “optimum pH”. the line represents the expected plot of the graph in the experiment. Effect of pH on Invertase/Enzyme Activity pH plays an relevant role in enzyme activity. generally. Enzymes perform well at pH 3-7 while beyond those values meant denaturation and loss of enzyme activity.Figure 3.054 0. those of extreme high pH value will mean. an ionic bond can either be formed or broken. pH Absorbance at 540 nm 1 3 5 7 9 11 0.the most favourable pH for an enzyme. This denatures enzymes. Values below the optimum pH will mean a slow reaction rate for enzyme. failure to completely comply with the procedures B. Thus results a distorted protein. lower and higher pH values than the optimum pH (Lower pHwhich describes the slow reaction rate for enzymes before achieving optimum pH. inaccurate measurement of the samples b. When this happens. From the results obtained by the group below.
Glucose Assay by DNS Method http://faculty.about.pdf 4.net/moodle/mod/resource/view.ksu.html Manual of Clinical enzyme Measurement.php? id=44 http://www.com/doc/58234422/Formal-ReportExperiment-3-Enzymes 5.scribd.worthingtonbiochem.edu.com/de/nestsite/modbio05. Biology:How Temperature Affects Enzymes http://www. Introduction to Enzymes http://www.angelfire. 1972. http://www.pdf 3. .woisd. Enzymes http://biotech.com/introBiochem/Enzymes. Philipps.0 REFERENCES: 1.sa/aabulhamd/Documents/II%20 lab/GLUCOSE%20ASSAY%20(353).com/od/glossary/g/Enzyme. Theresa.Figure 4.htm 2.
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