Question

We will assay enzyme activity at 340 nm. The molar absorbance is higher at 260, meaning we can detect three times less cofactor. Why don’t we use that wavelength? If you had to pick the worst possible wavelength to monitor, what would it be more context: doing assays after purifying proteins from animal tissue. Testing for enzymatic activity

Answer 1

One of the methods of testing or determining enzymatic activity is spectrophotometric enzyme assays. During a spectrophotometric enzyme assay an operator follows the course of an enzyme reaction by measuring the changes in the intensity of the light absorbed or scattered by the reaction solution.

If an enzyme activity is assayed at 340nm and molar absorbance is higher at 260nm, it means that at this wavelength the enzyme activity is higher. An ideal wavelength should clearly indicate the difference between the reactant and product with no interference from other chemicals in the reaction mixture.

In this case as absorbance is higher at 260nm we cannot use this wavelength as plotting of graph to calculate enzyme activity (intensity of absorption vs wavelength) would be difficult as this would not result in a normal curve.

The worst possible wavelength to monitor this enzyme activity would be between 200nm to 300nm to get a normal curve in a graph plotted with intensity light absorbed versus wavelength.

We can make use of the Beer's Lambert law to calculate the ideal wavelength as follows:

First calculate the concentration of the substrate using Beer's Lambert law : C = A / El

A=Absorbance of solution at a particular wavelength

C = Concentration of substrate

E = Molar absorbivity

l = Length of solution Cell

Once the concentration of the solution is obtained at different wavelengths, calculate Enzyme activity.

Enzyme activity = Moles of substrate converted per unit time.

Finally from enzyme activity calculation choose the wavelength range between which the enzyme activity is highest.