Enzyme Assay Lab Report

Enzyme assays are performed to serve two different purposes: (i) To identify a special enzyme by proving its presence or absence in a distinct specimen. (ii) To determine the amount of the enzyme in the sample by monitoring the disappearance of substrate or appearance of product. Enzymes speed up reaction rate by decreasing the activation energy required to start the reaction. Activation energy is the energy required to break certain bonds in the substrate so that other bonds can form. The formation of these new bonds results in the formation of the product by measuring the changes in absorbance due to the substrate (starch) being changed into product by the amylase enzyme. Since the starch and buffer used in the experiment contain chemicals

The activities of most enzymes follow a bell-shaped curve, increasing from zero in the strong acid region up to a maximum value which is identified at the optimum pH. Enzymes display their highest activity at their respective optimum conditions as seen in figure 2, the optimum pH is 6.5 since it has the highest absorption peak on the curve, and then decreasing to pH 8.5 which is the the strong alkaline region (Figure 2). The state of protonation is responsible for this behaviour, the protonation of one functional group promotes the catalytic activity, while protonation of another essential group breaks it down. In this case two conventional titration curves, an increasing and a decreasing one, form the bell-shaped curve as seen in figure 2.

A pH balance of 0-6 is acid, a pH balance of 7 is neutral, and a pH balance above 7.5-14 is alkaline. Buffers serve to adjust and stabilize the desired pH during the enzyme assay. They consist of a weak acid and a strong basic component. The buffers were incorporated into the starch to maintain the pH at their respective levels. The pH used in the practical varies from 5, 6, 6.5, 7, 7.5 and 8. At pH 7 is neutral and human intracellular enzymes work best at this pH, but in the practical performed we found the pH of the enzyme to work best at a pH of

If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or an enzyme might become inactive and denatured. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. Several factors are influenced directly by the pH in which the reaction takes place. Extremely high or low pH values generally result in complete loss of activity for most enzymes. Increased acidity or alkalinity decreases the ability of the substrates to bind to the active site and so enzyme action decreases, a major pH change denatures the enzymes so enzyme action stops. As we can see in figure 2, the increased acidity and alkalinity have lower absorbance values from the spectrophotometer than the absorbance value obtained at the optimum pH, due to lack of catalytic activity. Hence we can conclude that the digestive enzymes found in the salivia, such as the α-amylase, possess a pH optimum near the physiological pH of 6.5-