Hello Abu,

Mostly, enzyme activity based on spectrophotometry makes reference to the concentration and absorbance of a standard. What I mean by standard is a chemical that is mimicry of your expected product.

For example, in my experiment to determine the cellulose-degrading ability of beta-glucosidase (it's a cellulase), I use p-Nitrophenyl β-D-glucopyranoside as substrate(pNPG) and p-Nitrophenyl (pNP, normal exhibits yellow colour) as standard. In this case, the enzyme cleaves the bond between the p-Nitrophenyl and D-glucopyranoside (referring to the substrate) and, thus, showing the yellow coloration whose absorbance can be measured at a given wavelength (say, 400nm). 

This is how you could go about it in such a case:

Amount of product(pNP) yield = [conc of standard]/absorbance of standard)*Absorbance of reaction mixture

Note: the amount of product yield has the same unit as the conc of standard.

Enzyme activity= (Amount of product yield/time of reaction)

On the other hand, the specific enzyme activity is relative, and it varies based on ones definition. You can look at it in terms of mass of enzymes, optical density (OD) of enzyme bearing cells, etc.

In such a case, you just need to divide the enzyme activity by either the mass of enzyme used or the final OD in the reaction mixture.

It has a unit in the form of conc/time.mass or conc/time.OD.

Sorry for the lengthy talk, but I hope I made enough sense to you. All the best. 

Hello Abu,

Mostly, enzyme activity based on spectrophotometry makes reference to the concentration and absorbance of a standard. What I mean by standard is a chemical that is mimicry of your expected product.

For example, in my experiment to determine the cellulose-degrading ability of beta-glucosidase (it's a cellulase), I use p-Nitrophenyl β-D-glucopyranoside as substrate(pNPG) and p-Nitrophenyl (pNP, normal exhibits yellow colour) as standard. In this case, the enzyme cleaves the bond between the p-Nitrophenyl and D-glucopyranoside (referring to the substrate) and, thus, showing the yellow coloration whose absorbance can be measured at a given wavelength (say, 400nm). 

This is how you could go about it in such a case:

Amount of product(pNP) yield = [conc of standard]/absorbance of standard)*Absorbance of reaction mixture

Note: the amount of product yield has the same unit as the conc of standard.

Enzyme activity= (Amount of product yield/time of reaction)

On the other hand, the specific enzyme activity is relative, and it varies based on ones definition. You can look at it in terms of mass of enzymes, optical density (OD) of enzyme bearing cells, etc.

In such a case, you just need to divide the enzyme activity by either the mass of enzyme used or the final OD in the reaction mixture.

It has a unit in the form of conc/time.mass or conc/time.OD.

Sorry for the lengthy talk, but I hope I made enough sense to you. All the best. 

Dear Obeng

Thank you very much for your explanation. Could you please calculate specific activity from my above data and please let me know the results?

An important point to establish prior to calculating specific activity is whether the reaction proceeded at a constant rate during the time period prior to measuring the absorbance change.

As for the calculation, it has been answered many times on ResearchGate. Please use the search tool to find some examples.

Dear Md. Abu

Please find below formula which I had used 

Abs of Sample X Vol of reaction mix X 1000

---------------------------------------------------------

Molar Extinction of Std X Vol of enzyme in mL X time in minutes

This gives  activity in terms of  micromoles of NADH per ml per min.

Activity Units /Proteins in mg  = Sp activity U/ mg

Hope this helps you.

• @Trupti Ashar 
• could you please give me the refrence for these calculations???