Effect of Ph and Temperature on Enzyme Activity in Skeletal Muscle
Introduction:
The purpose of this experiment is to measure the effects of changes in temperatures and pH on enzyme activity in skeletal muscle, particularly the activity of lactate dehydrogenase (LDH). LDH is a glycolytic enzyme which converts pyruvate to lactate in the following equation: LDH Pyruvate+ NADH ------------ Lactate + NAD
The reaction above can move in both directions, forward (favored by Type II skeletal muscle) and reverse (favored by Type I skeletal muscle and the heart). Enzyme activity is important in this reaction because enzymes act as catalysts, they decrease the activation energy (the difference between the transition state and the energy of the reactants or products) needed to start the reaction and therefore speed up the reaction rate. A substrate molecule must bind to the enzyme at the active site before the substrate can be catalyzed. The rate at which an enzyme can catalyze the reaction is affected by factors such as enzyme concentration, substrate concentration, the affinity of the enzyme for the substrate (how well the enzyme binds to the substrate), concentrations of cofactors and coenzymes, pH and temperature.
In this experiment, we will be measuring enzyme activity in samples of muscle homogenate (skeletal muscle that has been broken up) by measuring how much light is absorbed by NADH in the above equation, which will be related to how much NADH is present. We will then determine LDH activity by using the formula: ([ A/min]/6.22) x 78,780, where [ A/min]=(initial absorbance-final absorbance)/3. We will be testing the absorbance using a spectrophotometer, which simply generates light that passes through a filter that causes the light to have a specific wavelength. NADH absorbs light maximally at a wavelength of 340 nm. Therefore, the measurement of NADH degradation is used to monitor the rate of lactate formation and
The purpose of this experiment is to measure the effects of changes in temperatures and pH on enzyme activity in skeletal muscle, particularly the activity of lactate dehydrogenase (LDH). LDH is a glycolytic enzyme which converts pyruvate to lactate in the following equation: LDH Pyruvate+ NADH ------------ Lactate + NAD
The reaction above can move in both directions, forward (favored by Type II skeletal muscle) and reverse (favored by Type I skeletal muscle and the heart). Enzyme activity is important in this reaction because enzymes act as catalysts, they decrease the activation energy (the difference between the transition state and the energy of the reactants or products) needed to start the reaction and therefore speed up the reaction rate. A substrate molecule must bind to the enzyme at the active site before the substrate can be catalyzed. The rate at which an enzyme can catalyze the reaction is affected by factors such as enzyme concentration, substrate concentration, the affinity of the enzyme for the substrate (how well the enzyme binds to the substrate), concentrations of cofactors and coenzymes, pH and temperature.
In this experiment, we will be measuring enzyme activity in samples of muscle homogenate (skeletal muscle that has been broken up) by measuring how much light is absorbed by NADH in the above equation, which will be related to how much NADH is present. We will then determine LDH activity by using the formula: ([ A/min]/6.22) x 78,780, where [ A/min]=(initial absorbance-final absorbance)/3. We will be testing the absorbance using a spectrophotometer, which simply generates light that passes through a filter that causes the light to have a specific wavelength. NADH absorbs light maximally at a wavelength of 340 nm. Therefore, the measurement of NADH degradation is used to monitor the rate of lactate formation and