As a substrate of the enzyme, ascorbate might be there to help stabilize it during purification.

Dear Prof. Panda,

Ascorbate peroxidase is a central component of ascorbate gluathione cycle, and plays an essential role in the control of intracellular ROS levels. APX uses two molecules of AsA to reduce H2O2 to water with a concomitant generation of two molecules of MDHA. This happens possibly due to the electron donated by the ascorbate (AsA). In this way, it also stabilizes the APX.

I think this could be a possible explanation of your query.

Best regards,

Amit

We do not use ascorbate in the extraction/homogenizing/grinding buffer and I suppose we have been getting good activity of ascorbate peroxidase even in our crude enzyme extracts. Use of ascorbate in extraction buffer might have negative effect as the enzyme would start showing its optimal (?) activity (as hydrogen peroxide would also get extracted in the buffer) during homogenization itself. If turnover rate of this enzyme is high, it might even loose its activity by the time one starts assaying. I do not know turnover rate of ascorbate peroxidase. In live cells, substrates would increase expression/over-expression (i.e. synthesis) of concerned enzymes. However, to the best of my knowledge during grinding/homogenization I do not expect any enhancement in expression (i.e. transcription-translation and post-translational alterations).

I shall be highly obliged if Prof. Adam Shapiro and other experts can comment on this aspect and also on mode of probable stabilization of ascorbate peroxidase by ascorbate.

Stabilization of enzymes by substrate binding is a well-known phenomenon and is often used during protein purification.

In the absence of peroxide during purification, there will be no turnover of added ascorbate by the enzyme. If there were peroxide present when the extract was made, the peroxide would be destroyed by the action of ascorbate peroxidase if ascorbate were present. This would be beneficial to maintaining the activity of the enzyme, since reactive oxygen species such as peroxide are damaging to proteins.

Once cells are ground up, the dilution of the cytoplasmic components necessary for protein translation stops any further protein production, so induction of ascorbate peroxidase production by addition of ascorbate during extraction will not occur.

While I thank the respected researchers for their meaningful inputs to my query, I do endorse the explanation provided by Prof. Shapiro. I am now convinced that ascorbate is added to the grinding buffer to help stabilization of the enzyme.

Dear Prof. Adam Shapiro,

Thanks for your excellent clarification. Can you please suggest one or two recent publications on stabilization and/or protection of enzymes by substrates?

From your clarification, it is also clear that it is better to have an ideal scavenger of ROS in extraction buffer (like the way we use EDTA to chelate metal ions, cysteine/DTT/mercaptoethanol to prevent unnecessary disulphide bond formation between free cysteine of different proteins/polypeptides, PVP/PVPP to chellate phenolics etc. in extraction buffer). Can you please let me know, if anyone has used ROS scavenger in extraction buffer and can you please suggest some ideal scavenger of ROS that can be used (ofcourse without interfering with activity of any enzyme that one wishes to assay)?

 It's slightly off-topic I know, but I like this recent paper by one of my colleagues, who found that expressing an enzyme in the presence of an inhibitor stabilized it to the extent that it's solubility during overexpression was greatly increased.

http://www.ncbi.nlm.nih.gov/pubmed/25240855