Purpose: The aim of this experiment is determining Km by using three methods which are Michaelis Menten equation, Eadie Hofstee and Lineweaver Burk plot.
Materials: Double beam spectrophotometer, test tubes, pipettes
Enzyme solution: 10 mg/ml enzyme in distilled water
Substrate solution: p-Nitrophenyl acetate in acetonitrile
Buffer: 200 mM potassium phosphate buffer, pH 7.4
Procedure: First of all, according to table below the reagents was diluted into spectrophotometer cell at each substrate concentration for each step in the table. And then the change in absorbance with time was followed by a double beam spectrophotometer for each substrate concentration in the table. However time versus absorbance graph for each substrate concentration was printed by spectrophotometer. From this graph initial rate versus substrate concentration was found. And finally from obtained data mK methods. value of the enzyme substrate reaction values was found by using three