Enzyme Kinetics Essay

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Enzymes are catalytic proteins that are essential in human metabolism, as they help facilitate many of the biochemical reactions occurring in the body by lowering the activation energy that is necessary for each reaction to occur. Various factors, such as temperature and pH, contribute to the enzyme activity and can be directly regulated to affect the overall rate of reaction. The purpose of this experiment is to determine the optimal physical conditions of the glyocolytic enzyme muscle lactate dehydrogenase (mLDH), which aids in the reduction and conversion of pyruvate to lactate and the oxidation of NADH in skeletal muscle, and to discover the effects of altering temperature and pH on the activity of the enzyme mLDH. Methods In order to complete this experiment and determine the optimum pH and temperature of the enzyme mLDH, 16.2 mM pyruvate in dH2O was placed in 0.2 mM NADH in 54 mM phosphate buffer solutions of varying temperatures and pH levels, in accordance with the following reaction: pyruvate + NADH + H+ lactate + NAD+ In this experiment, 900 µL of NADH solution with a pH 5.5 (at about 25°C) and 25 µL of 1:2000 dilution of muscle homogenate (containing mLDH) were mixed inside a cuvette. After waiting a minute, 50 µL of pyruvate was then added to the cuvette and then mixed. The cuvette was then placed in a spectrophotometer and the initial absorbance of the solution was measured. Subsequent absorbance measurements were taken each minute over a three minute period. The overall enzyme activity was measured indirectly through the absorbance of the solution. Absorbance was measured using a spectrophotometer, an instrument that measures the light absorption of a solution at a given wavelength with a spectrophotoelectric cell. Before each absorbance measurement was taken, the spectrophotometer was zeroed at 340 nm by a cuvette filled with deionized

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