Although enzymes have different pH optimal for activity, they will still have some level of activity at a nearby pH, so you should be able to make a measurement. Moreover, if you are going to dilute the extract into the reaction mixture for each assay, the pH will be closer to the reaction mixture's pH, assuming it is buffered at a similar strength. If the dilution factor is large, the pH will be essentially the same as the reaction mixture. For example, if you dilute your pH 7.5 extract 1:100 into a PAL reaction mixture buffered at pH 8.8, the pH will be pH 8.8. So make sure the substrate for each assay is prepared in a buffer at the optimal pH for that assay.

Hi Bellamkonda

First I don't know if you are using the same substrate in the assay for all enzyme or not, if so then you cant use this extract due to interfere that may occurred, secondly bear in mind that many enzymes have broad pH i.e some work between pH 6-7.8 so in your case you will not be able to say which enzyme has metabolised the substrate, my advice to purify the one you want and then assay to be in safe side or you could add inhibitor (if known) to inhibit some.

If these data for publication then I recommend to purify each one then assay.

If these enzyme have different molecular weight then try gel filtration to separate them

Hope this helps & good luck

Hi Bellamkonda

I think that if you use an aliquot of the enzymatic extract in the reaction mixture buffered and guarantee that is ideal pH for enzymatic activity.

Sincerely

It is better to lyophilize the crude enzyme extract so that you can have concentrated enzyme source for enzyme estimation by keeping the respective reaction conditions ( pH , temp.; time ) and protein concentration optimal otherwise the crude enzyme will be to dilute to get  good reproducible activity

What is your substrate?  I think you are saying there are three enzymes in one extract and you wish to run assays at differentpH's to optimize each actvity...is that correct?  Can you separate the three enzymes by  HPLC or other means?  Will you be using the same three enzyme combination in the future?  Can you reproduce that extract? 

You can use each substrate at proper pH with higher buffer concentration than that of pH 7.5 PB. For example, If pH 7.5 buffer at 50 mM, you can use 100 mM buffer containing hydrogen peroxide at pH7 for CAT activity. Similarly, for POD activity, 150 mM pH 6.5 buffer solution which conatins POD substrate.