All data were collected at Dickinson College
Beta-galactosidase (bGal) is an enzyme that cleaves polysaccharides containing galactose to liberate galactose; for instance, lactose is cleaved by bGal into glucose and galactose. In this experiment, kinetic properties of E. coli b-galactosidase with three different potential inhibitors, IPTG, L-arabinose, and theophylline were studied using hydrolysis of ONPG to release o-nitrophenol.
Each reaction set (performed in triplicate) contained 200 microliter solutions, included 50 microliter of ONPG as substrate solution (with varied concentrations), 50 microliter of reaction buffer (for uninhibited reaction) or potential inhibitor solution (IPTG/L-arabinose/theophylline), and 100 microliter of bGal solution.
O-nitrophenol released from the reaction formed p-nitrophenolate ions, whose absorbance can be measured at 420 nm using UV-Vis spectrometry. Measurements were read every 45-60 seconds in around 7 minutes. Reaction rate (v) with substrate solutions of varied concentrations ([S]) were used to generate Michaelis-Menten and Lineweaver-Burk plots for kinectic analysis.