Abstract
The study of enzymatic catalysis is a classical biochemistry experiment for undergraduate classes. We propose the utilization of the serine protease trypsin to discuss several parameters affecting enzyme catalysis. Hydrolysis of the chromogenic substrate Nα -benzoyl-arginine-p-nitroanilide (BApNA) was followed by spectrophotometric monitoring. The optimal pH and temperature values were found to be 8.0 and 40 °C, respectively. Km and Vmax values were obtained by adjustment to Michaelis-Menten, Lineweaver-Burke, and Hanes equations. We then investigated the effect of organic solvents (a series of alcohols) on the hydrolysis of the chromogenic substrate. The reaction rate was reduced in the presence of methanol and further reduced by ethanol, 1-propanol, and 2-propanol, when compared to the data obtained with buffer. Finally the students were asked to measure the molar absorptivity of p-nitrophenol in the presence of the alcohols employed for the kinetic experiments. Thus they could learn that the value of t...
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