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However different concentration of o- Interchangeableness (0. 3, 1. 5, 2. 1, 2. 7, 3. 0) is used with the same incubation temperature of ICC. Spectrophotometer is then used to find out the absorbency against time over minutes. For this experiment, the sample with concentration of 3. 0 has the greatest rate of increase in absorbency and also the highest absorbency reading of 0. 022 at the end of min. Thus it has the highest enzyme activity. Objective: The purposes of experiment 5 are to let student familiarize the usage of spectrophotometer, handling and using the microcomputers and to study the effect of enrapture on enzyme.
The purposes of experiment 6 are to allow student to understand the practical aspect of handling enzyme, to study and measure enzyme activity and how concentration of substrate affects enzyme activity. Introduction: Spectrophotometer Spectrophotometer is a method to measure how much light can a substance absorbs by measuring the intensity of a light beam passing through a sample solution using spectrophotometer. The principal is that each compound absorbs certain range of wavelength and this can be used to measure the amount of a known substance. Beer-Lambert Law
Beer-Lambert Law states there is a linear relationship between absorbency and sample and is commonly used in chemical analysis. Thus, Beer-Lambert Law can be only used when linear relationship is present. The Beer-Lambert law can be obtained from an approximation for the absorption coefficient for a molecule by estimating the molecule using an opaque disk whose cross-sectional area (o), represents the effective area seen by a photon of frequency (w). If the frequency of the light is far from resonance, the area is about O, and if w is close to resonance the area is maximum.
Beer-Lambert Law written as: A-??LLC A: the measure of absorbency (A = logo POP / P) E: Molar absorptive (L mol-l CM-l as unit) l: path length (CM as unit) c: concentration (mol L-1 as unit) Reaction between o-Indistinguishableness (GNP) and Beta-Glaciological GNP have similar structure as lactose and is used as a false substrate known as a analog, the enzyme does not differentiate between them and cleaves the Beta-IA glycoside linkage with an addition of an water molecule to form calaboose and o- nitrogen.
The reason for using GNP as substrate is GNP is colorless but the reduce o-nitrogen (NP) is yellow and can be measured by spectrophotometer. By measuring the rate of color change, enzyme activity can be measured. The more yellow the sample, the more GNP has been hydrolysis, higher the absorbency indicating a greater enzyme activity. Enzyme Kinetics Enzyme kinetics is the study of chemical reaction of enzyme catcalled reaction and how the rate are affected with the change in experimental conditions. Rate of reaction is measured and enzyme is exposed to various conditions and the effects are observed.
This way, the enzyme’s catalytic mechanism, metabolism role, how its activity can be controlled can be known. Enzyme, usually a protein molecule that affects other molecule (substrate), there’s substrate binds to an enzymes’ active site forming the enzyme-substrate complex is later hydrolysis into its products. Procedure: Experiment 5 Experiment 6 Result: Temperature ( C) 0. Mm Absorbency (NM) Mm Absorbency (NM) Control 25 0. 01 0. 035 0. 025 0. 069 50 0. 023 0. 071 0. 016 0. 04 70 0. 006 0. 027 A control is used to ensure accurate result.
From the graph, the optimum temperature of the enzyme for 3. Mm substrate is 50 co Experiment 6 Absorbency vs. Time Concentration . 3 Time 0. 004 0. 003 0. 005 10 0. 007 0. 008 20 30 1. 005 ). 005 ). 007 1005 0. 010 0. 011 55 0. 009 0. 012 65 0. 013 75 0. 014 85 0. 01 1 0. 015 95 105 110 115 120 0. 017 125 130 0. 018 135 140 0. 019 150 155 0. 020 160 165 170 0. 021 175 180 0. 022 All the concentrations have a similar trend, absorbency increases with time. However, the rate of increase and the final absorbency is greatest for Mm, thus it has the highest enzyme activity.
Discussion Interpretation of result Experiment 5: This experiment serves the find out the effect of temperature on enzyme. For experiments with 0. Mm and Mm substrate, the enzyme activity increases with increasing temperature and peaks at ICC and ICC respectively, the optimum temperature where the most enzyme activity occurs and then declines as the temperature continue to increase. This reaction is exothermic, the sample with greater concentration will produce more heat due to more collisions between reacting molecules, thus 3. Mm substrate have greater enzyme activity and optimum temperature than 0. Mm substrate. Both graph shows a similar trend, increases in enzyme activity and peaks at a point and then decreases in enzyme activity with increasing temperature. This can be explained as the temperature increases, the enzyme and substrate gain kinetic energy and moves faster and result in more effective collision, thus more substrate and enzyme bind to form enzyme-substrate complex, as a result more product are formed and formed in a faster rate.
The reaction is fastest at optimum temperature as the enzyme and substrate are moving at a rapid speed without any denomination occurring. Any further increase in temperature beyond optimum temperature cause the rate of reaction to decrease. This is due to the denomination of enzyme which is irreversible. With the active site of the enzyme altered, which is complementary to substrate. The substrate can no longer bind to the active site thus enzyme substrate complex cannot be formed and no products will be formed.
As the temperature increases beyond the optimum point, more and more enzymes are denatured and it will reach a point where all enzymes are denatured, thus no reaction can take place. Other similar experiment conducted by Biomedical have results of Beta-glaciological having an optimum temperature of ICC. Which is the exact or close value I obtained from this experiment, thus I can justify that my experiment is accurate and successful.
Experiment 6: This experiment serves to find out the effect of concentration on the rate of enzyme activity at a specific temperature, ICC. All the graphs shows a similar trend of increasing absorbency with time, indicating an increase in enzyme activity. As time progresses, GNP substrate and enzyme is reacting and more products are being formed in this reaction. This can be seen as o-Nitrogen, a product released at the end of the reaction, which is yellow in color can be used to measure to absorbency. Thus, higher absorbency indicates more product formed.