I am currently doing an experiment and working on amylase, lipase and protease enzyme activity. I have followed various protocols and have done experiments. I am looking for assistance in doing the calculations for the enzyme activity. Anyone with said expertise, I would appreciate your assitance.
I could just give a formula but I think more explanation is helpful when you are new to enzyme activity.
First you need to define your enzyme unit. A typical definition for a unit would be the amount of enzyme catalysing the transformation of 1 umol of substrate per min under standard conditions (I.e. your conditions to be pragmatic). Typically you would measure appearance of product and not loss of substrate, and the stated definition works fine if one substrate molecule gives one molecule of measurable product.
Presumably you have a standard curve showing the relationship between signal (y) and product concentration (x)? For your samples, determine the x values from the measured y values. From those, determine the umol of product formed. This is simple if you plotted umol on your x axis. If you plotted concentration, you’ll need to work out umol before proceeding further.
Once you have the umol value, divide by your assay time, to give umol per min. I.e units. Activity is usually expressed as units per ml, hence divide this number by the volume of your assay in ml, to give units per mL OF YOUR ASSAY MIXTURE. If you want activity of your stock enzyme you’ll need further multipliers. E.g. If the enzyme is 20% of the assay volume, multiply the activity by 100/20. If you diluted the stock enzyme 1/100 before adding it to the assay, then in that case you would also multiply by 100 to get units per ml of neat enzyme stock.
I hope this helps.
THERE ARE A SERIES OF VOLUMES IN METHODS OF ENZYMOLOGY
also
TitleMethods in Enzymology Volume 61 of Enzyme Structure, Serge N. Timasheff Enzyme Structure: Part H, Serge N Timasheff Methods in Enzymology, ISSN 0076-6879 Volume 61 of Methods in enzymology: Enzyme structureEditorsC. H.W. Hirs, Serge N. TimasheffContributorsC. H.W. Hirs, Serge N. TimasheffEditionillustratedPublisherElsevier Science, 1979 and
Rossomando EF (1990) Measurement of enzyme activity. Methods in Enzymology 182: 38–49
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