Spectrometric Assay Lab Report

Natural enzymes are proteins that catalyze biological reactions by lowering the activation energy of the reaction without being altered during the process. The enzyme used in this experiment was the β-galactosidase purified from E. coli. This enzyme hydrolyzes lactose and turns it into galactose and glucose. Since it is difficult to assay the activity of β-galactosidase, we will be using the artificial substrate, o-nitrophenyl-β-galactoside (ONPG) instead of lactose. ONPG is an analog of lactose and an advantage of using ONPG is that it is easy to determine the amount of ONPG cleaved by using spectrometric assay (1). The β-galactosidase hydrolyzes ONPG and yields a yellow solution that contains o-nitrophenol and galactose. The solution becomes more yellow as the more ONPG is being degraded. Using spectrophotometry, the absorbance of the solution can be determined at a wavelength of 420nm. The assays will help determine the Km, Vmax, and Kcat of the enzyme. In our assays, Na2CO3 is used to stop the reactions by changing the solution pH to basic and as a result the enzyme will become inactive.
The
The Michaelis constant, Km equals to the substrate concentration when the rate of the reaction is ½ Vmax. As the concentration of enzyme increases, all substrates will be used up; as a result the rate of reaction reaches a plateau (Vmax). Another way to determine the important parameters is to convert the Michaelis- Menten equation into the y = mx+ b form. Taking the reciprocal of the Michaelis- Menten equation gives an equation that forms a straight line, which is called the Lineweaver- Burk equation. The parameters can then be extracted from the y- intercept (1/ Vmax) and the slope (Km/ Vmax). The Kcat can be found by dividing Vmax by the enzyme concentration. The equation below is the reciprocal of the Michealis –Menten