If i want to perform any enzyme activity how to select the substrate concentration in 3 mL reaction mixture? and if there is any donor is present in 3 mL mixture how to select the concentration of donor? What is the logic to select these components in 3 ml reaction mixture? Please suggest me to do the activity assays/
I would like to recommend the HPLC-photometric and/or HPLC-fluorimetric enzyme assay. The volume of the reaction solution is reduced to 0.1 mL (please see the file J Chrom B ,,,). Further, I must add the comment that only HPLC-photometric enzyme assay gives linear-calibration line inserting to zero point, and stoiciometric due to purification by HPLC; i.e., the other photometric methods are only measuring the product , and non-linear determination of the product due to non-separation (crude state) or non-purification.
Hi
An enzyme accelerate (speed up) reaction, but the substrate concentrations may also have effect on how fast this reaction is, for this reason you need to do a simple time course (several time points) experiment by incubating x amount (units or mg) of the enzyme with different substrate concentrations. This should gives you an idea about what's substrate concentration should be used, incubation time.
Refer to the following link although its for catalase but useful.
http://schoolworkhelper.net/what-effects-of-concentrations-of-a-substrate-on-enzyme-lab-answers/
I suggest to read about enzyme kinetics, surely you'll find the answer you looking for in any biochemistry text book.
Hope this helps
What enzyme that you are dealing with? The assay varies with the enzyme types.
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