B-Galactosidase Lab Report

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1.1 Abstract

The purpose of quantitative analysis of protein using a spectrophotometer is to measure the concentration of proteins in a given sample. The experiment is conducted by laboratory method (Biuret Test) and using spectrophotometer to analyze the absorbance of reactants at 540 nm, hence determining the concentration of the proteins in a given sample.

The purpose of stopped enzyme assay to study B-galactosidase is to determine the effect of temperature and concentrations of substrate on enzyme activity. B-galactosidase breaks down the disaccharide lactose into simple sugars glucose and galactose. However, glucose is a colorless compound hence it has to be substituted with a compound that is detectable by a visible color change. Hence, o-Nitrophenylgalactoside (ONPG) is used to replace lactose. Upon hydrolysis by B-galactosidase, the colorless ONPG produces galactose and the substitute yellow colored compound o-Nitrophenol (ONP). The change in color intensity is measured by the spectrophotometer at 420 nm, hence determining the enzyme activity after all activities have come to a stop.

The purpose of continuous enzyme assay to study B-galactosidase is to determine the rate of enzyme action (B-galactosidase) at different substrate concentrations of o-Nitrophenylgalactoside (ONPG). The experiment is conducted by adding of the enzyme into the substrate preparation quickly and measure the change in absorbance of the reactants as a function of time. By plotting the

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