I am working on a protease enzyme and recently i have decided to perform a pH optimization as well as temperature optimization for maximal activity of the protein using azocasein as substrate.
Somehow i am getting little bit confused in case of pH optimziation. I have no idea how to maintain different pH for activity because if i will add the azocasein and enzyme solution in a buffer have desired pH it will certainly change that one a little bit.
It would be great if someone can provide me a detailed protocol to perform it and help me out in this.
Prepare reaction buffers using a set of buffers with overlapping pH ranges. For example, if the protease works best at near-neutral pH, you could make buffers with pHs in the range 6.5-7.5 with MOPS, 7-8 with HEPES, and 7.5 to 8.5 with Tris. Use at least 50 mM buffer to avoid pH changes. This approach allows you to identify not only the optimal pH, but also the optimal buffer. Here is a list of buffers and their useful pH ranges.
http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html
Hi,
In order to have minimal effect on the pH you should have the enzyme in the desired pH buffer, made as suggested above(I would also suggest to have 100 mM NaCl added in all buffers to keep the ionic strength constant because it affects enzyme activity) and make the dillutions in a way that the enzyme will be in the 90% of the final volume and the substrate will be 10fold concentrated (from the desired final concentration) but has only 10% of the final volume. This way the substrate buffer will have minimal effect on the final pH upon mixing and starting of the reaction. Good luck!
@vlatko and @Adam Thanks for your suggestions i am going to make my experimental protocol following these guidelines and will perform experiment next week. Hope it will work.
Hello Vishal,
To optimize the pH for the activity of the said protease, carry out the standard protease activity Assay using different buffer solutions (i.e change only the pH of the reaction) especially around the pH range at which the enzyme activity is high. However, be mindful of the buffer to be used at particular pH range. For instance, Acetate buffers (pH 4.5-5.5), Phosphate buffers (pH6-7.4), Tris-HCl (8-9). To determine the pH stability, pre-incubate the enzyme in solutions of relevant buffer for a time determined by you, let say 2 hours while making periodic withdrawals of the aliquot (maybe at 20 min intervals) to be used as enzyme solution in the standard enzyme activity assay. With this, you can calculate the residual activity of the enzyme and determine how stable it is at each pH.
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