B-Galactosidase Lab Report

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,

Enzymes speed up chemical reactions enabling more products to be formed within a shorter span of time. Enzymes are fragile and easily disrupted by heat or other mild treatment. Studying the effect of temperature and substrate concentration on enzyme concentration allows better understanding of optimum conditions which enzymes can function. An example of an enzyme catalyzed reaction is enzymatic hydrolysis of an artificial substrate, o-Nitrophenylgalactoside (ONPG) used in place of lactose. Upon hydrolysis by B-galactosidase, a yellow colored compound o-Nitrophenol (ONP) is formed. By using a spectrophotometer to measure absorbance at 420 nm, the rate of enzyme activity after all reactions have come to a stop can be

However, all proteins are constructed from the same set of 20 amino acids linked in unbranched polymers. The covalent bond that exists between amino acids is called peptide bond, hence a polymer of amino acids is named polypeptide. A protein is a biological functional molecule made up of one or more polypeptides which is folded and coiled into unique three-dimensional structure.

In laboratory, it is important to measure the concentration of proteins for research investigations. Biuret test is adopted to quantify proteins in fluid by using a spectrophotometer. The biuret solution is a blue solution made up of sodium hydroxide and copper (II) sulfate which turns pink or violet in the presence of proteins, peptides and compounds containing 2 or more peptide linkage. A spectrophotometer measures the respective amounts of light consisting of different wavelengths absorbed and transmitted by a pigment solution. Spectrophotometer works when white light is separated into lights of different wavelengths by a prism which different colors of light passes through the sample. The transmitted light strikes a photoelectric tube, converting light energy to electric current that is measured by a galvanometer. The transmittance reading measures the absorbance of reactants and determine the concentrations of protein