|MadSci Network: Biochemistry|
That's not an easy question to answer. A number of things have to be considered in making and storing crude extracts, and what is important will vary with the enzyme(s) of interest and the specific tissue. The things you should probably first consider are:
1. pH - Remember that the pH for optimal activity of the enzyme may not be the pH for optimal stability. You may be able to get some idea of what you need from the literature, but the surest way is experimentally – store samples of extract at different pH values for a fixed time at 4 C, and then assay.
2. Any cofactors, reducing agents, etc. that are required for enzyme stability – again, you can probably get some idea of this from the catalase/peroxidase literature.
3. The presence of inactivating factors in the extract – especially proteases. Plant seed cotyledons can contain numerous proteases, both exo- and endo-peptidases. Proteases in the aspartic, cysteine, and serine mechanistic families are common. The inclusion of a protease inhibitor cocktail can improve the recovery and stability of other enzyme. Cocktail mixes inhibiting most protease families are available commercially (Sigma, Roche, Pierce, etc.), or you can add specific inhibitors singly. Note that protease activity is also pH dependent, so judicious choice of pH can also minimize inactivation of your enzyme by proteases. Another consideration in plant tissues is the presence of phenolics, which can also inactivate the desired enzyme. One can include polyvinylpolypyrrolidone in the extraction to bind these phenolics before they inactivate the enzyme of interest.
In the end, it really finally comes down to doing some pilot experiments testing pH, including inhibitors or not, etc.
Hope that helps.
Karl A. Wilson
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