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 therefore the LDH activity. Enzyme activity is important in skeletal muscle because the enzymes help speed up the reaction rate of anaerobic respiration, taking place in the body. Anaerobic respiration is a process of cellular respiration when there is no oxygen present. It converts the pyruvate from glucose molecules, to lactate to create ATP, which is a form of energy used by cells in the body. If the reaction is sped up by enzyme activity, then more ATP can be made in a shorter time period, and the cells can perform their work much quicker. Methods:
The equipment used in this lab experiment is as follows:
•Cuvette of deionized water
•3 Cuvettes with samples of muscle homogenate, each with 0.9 ml NADH at specific pH of 5.5, 7.4, and 8.5, and 0.05ml of pyruvate. •3 Cuvettes with samples of muscle homogenate, each with 0.9 ml NADH at specific temperatures of 0 C , 25 C, and 37 C, and 0.05ml of pyruvate. •Calculator
As stated before, the reaction we will be looking at is the following:
LDH Pyruvate+ NADH ------------ Lactate + NAD Where LDH is the enzyme we will be monitoring and NAD is the coenzyme. If the reaction goes forward, then pyruvate is the reagent and lactate is the product and if the reaction goes in the reverse direction then lactate is the reagent and pyruvate is the product.
To start, the spectrophotometer must be calibrated using the cuvette of deionized water to set the scale to zero. Then, the cuvette of muscle homogenate with NADH (pH of 5.5) is placed into the spectrophotometer, after being mixed, and the initial absorbance is recorded (at 340 nm). The cuvette is left in the spectrophotometer the absorbance is recorded after 1 minute, 2 minutes and 3 minutes. The cuvette is removed and the deionized water is used to set the spectrophotometer back to zero. Do this...