Understanding Enzyme Measurement Using ONPG Analog

Because it’s very difficult to accurately measure lactose or glucose in solution, we use a false substrate (known as analog) for the enzyme known as ONPG (ortho-nitro-phenyl-galactoside). ONPG has a structure similar to lactose, so it can bind to the enzyme and be cleaved. The products are galactose and ortho-nitrophenolate. ortho-nitrophenolate is yellow in color and can be measured by spectrophotometry. the rate of an enzyme can be measured. The reaction is usually expressed like this
E is the enzyme, S is the substrate, ES is the complex of the two and P is the product. Each step 1) formation of ES and 2) formation of product goes forward, as well as backwards. The backwards direction increases over time (as the reaction approaches equilibrium).

In order to make that correlation, you would need to measure a series of known dilutions of ortho-nitrophenolate, measure them with the spectrophotometer. the standard curve plotted Absorbance versus concentration for known amounts of ortho-nitrophenolate will be drawn. This will be useful in determining the rate of reaction for the Lactase enzyme in different conditions (amount of product/time = rate). D ifferent concentrations of different concentrations of ONPG is needed.
At this point the amount of lactase activity in your stock is unknown, so it would be good to determine a proper concentration of lactase to, therefore a series of dilutions of this first stock will be made and tested. Make a set of 4 serial 1:10 dilutions and label them as 1:10; 1:100; 1:1,000 and 1:10,000. This will give you a set of stock enzyme solutions of different concentrations.
After the working concentration of Lactase has been found, the two main measurements of enzyme kinetics need to be calculated: Vmax (maximum rate of reaction for this enzyme and this substrate under these conditions) and Km (the Michaelis constant, the concentration of substrate that gives one half Vmax – it’s an indirect measure of the affinity of the enzyme for the