Do you mean µg of product formed/mg of protein, or µg of enzyme/mg of protein? In the first case, you need to make a standard curve of product (OD versus µg) with which to compare the enzyme activity. In the second case, you need to measure the purity of the protein by some means, such as SDS-PAGE.

It will be easier for you to make a standard curve of absorbance against concentration separately and then extrapolate the concentration of the samples from the curve. Or if you used kits for your assay, normally a standard is provided and a given concentration of standard will be specified. Most often, the formular is: Conc. of sample = (OD of sample/OD of Std) * concentration of std.

See the link on similar work although the calculation wasnt specified.

Article Determination of Total Protein, Superoxide Dismutase, Catala...

Respected Adam B Shapiro, first I acknowledge for your concern. I mean the first one i.e., µg of product formed/mg of protein, but I have only the values of OD at specific wavelength, how to get values of µg to make standard curve?? Please narrate. Thank you again.

Dear Chukwuebuka Egbuna, First thank you very much for your effort. I want to know how to get the concentration to make standard curve. Please explain.

There are 2 ways to prepare the standards. The first way is to obtain the product that is to be measured and make a series of solutions covering the range of concentrations of interest in the experiment, then measure the absorbance (OD) associated with each solution. Sometimes, this method can't be done because the product is not available or is unstable.

The second method is to start from the substrate of the enzyme reaction. Prepare solutions with various concentrations of the substrate equal to the concentrations of product that you wish to have on the standard curve. Then add an amount of enzyme sufficient to convert all of the substrate to product in a reasonable amount of time. The absorbance changes that are measured at each concentration are then used to construct the standard curve.

Needless to say, the standard curve measurements must be made under identical conditions as the conditions in which the assay will be performed.

With the standard curve completed, it is now possible to convert any OD reading from the reaction into a concentration of product. Note that these are concentrations, such as µg/ml. To convert to µg, you have only to multiple by the sample volume. Then divide by the number of mg of protein in the reaction to get µg product formed/mg of protein.

Dear Syed,

In addition to the explanation provided by Adams, it is advisable to set up your excel worksheet or any other graphical software to display the equation of the standard curve and the correlation coefficient. With the equation, you can determine the concentration or the activity of the enzyme while the correlation coefficient (R) tells you the linearity of the graph by indicating if you serially diluted well. A good R value should be 0.99 or 1.

The major thing about it is that you prepare known concentration say 10, 20, 40, 60, 80, 100 of the standard SOD, which definitely when read will give correspondent absorbance. These absorbance obtained will be used to plot a graph against the concentration. The Y-axis represents Absorbance, while, the X-axis represents concentration. 

Best regards,

Egbuna, C.