Would it be correct, if at some fixed concentration of substrate increasing enzyme concentration till the enzyme attain saturation, and then by dividing the corresponding enzyme activity of a saturation point by the enzyme amount at that saturation point.?
Would it be appropriate, prior knowing the Km, can we calculate the specific activity of enzyme.?
Thanks.
If you can figure out the rate (units), then divide it by mg of your enzyme SA=U/mg
Dear Irshad Baig:
The idea you suggested of increasing the enzyme concentration is close, but I think you would do better to increase the substrate concentration until you didn't get a significant increase in observed enzyme activity, then divide that activity rate by the concentration of enzyme. Specific activity for enzymes isn't exactly defined perfectly enough to say there is a "standard" way of doing it for all enzymes, but you'll get the most commonly defined way by doing it as suggested above.
Usually, the substrate is cheap and easy to get and the enzyme is hard, but if it is the other way around (for some enzymes it is easy and inexpensive to get the enzyme and difficult and expensive to get the substrate or run the assay) - if this is the case then one usually picks a standard concentration for the substrate (example would be 1 microgram/L, or micromole/L, or whatever concentration would be expected in vivo) and calculate it using this turnover rate.
Good luck, and I hope this addresses what your question really was.
The specific activity is Vmax/enzyme concentration. If the enzyme has only one substrate, then it is usually only necessary to measure the rate at a saturating substrate concentration, then divide by the enzyme concentration. Care is needed to be sure that there is no inhibition at high substrate concentrations. if there is, Vmax can be determined by fitting the data to a kinetic rate equation that includes substrate inhibition. For multisubstrate enzymes, determining Vmax is more complicated. All the substrates should be titrated. If there is no substrate inhibition by any of them, then one can just use saturating concentrations of all substrates to get Vmax. If there is any substrate inhibition, it will be necessary to obtain Vmax by fitting the data to a kinetic rate equation. Note that the specific activity depends upon all the conditions of the assay (temp., buffer composition, substrate concentrations), which must be reported along with the specific activity to make it useful to others.
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