4/27/17

Dear Phoebe,

I will try to answer your questions. Many protease assay procedures use casein as the protein substrate. It's easy to get, inexpensive and of high purity.  At the end of the incubation period, TCA is added. This stops the enzyme reaction and denatures the casein, rendering it insoluble. The insoluble casein can then be removed by centrifugation or filtration to yield a clear solution. The protease reaction products in the clear filtrate  (e.g., amino acids, peptides)  can be visualized for quantitation purposes without interference by the casein. E.g., Tryptophan, phenylalanine and tyrosine can be detected by UV @ 280 nm. They and other AA's and peptides can also be detected with ninhydrin or o-phaldialdehyde (Roth reagent).

As far as the cysteine is concerned, my guess is that this is included in the assay procedure because many proteases (especially plant proteases) have a cysteine residue in their catalytic site that is required for activity. The added cysteine may serve to keep the cysteine in the enzyme's active site in the reduced (-SH) form.  See the  Wikipedia reference: https://en.wikipedia.org/wiki/Cysteine_protease.

I hope this information helps you. If you have other questions, feel free to contact me at your convenience. Good luck w/ your research.

Bill Colonna, Center for Crops Utilization Research, Iowa State University, Ames, Iowa, USA.  [email protected].

 Thank you, Colonna. Your answer is really helpful. 

 Wow =)). Thanks for your question @Phoebe and your answer Colona. 

Calla weonazo

Just remember though, that TCA concentration has an impact on the size of proteins/peptides to be precipitated. For example, after hydrolysis, 20% TCA would precipitate most of the short peptides hence free amino acids are soluble. When only 5% TCA is used, some short peptides are soluble. So, although it is an easy procedure, the method actually provides only quantification of the so-called TCA-soluble nitrogen (TCA-SN) and does not necessarily mean that whatever was hydrolysed by the enzyme was separated from the intact protein since some "poly"-peptides would be insoluble despite having undergone digestion.

Another thing to remember is that TCA must be used COLD. Hot TCA would hydrolyse the protein. So IF the temperature is NOT cold, one would better run a blank as well to ensure the "artefact" effect coming from TCA-digestion is NOT attributed to the enzymes (by mistake).

30% TCA How much Normality?

Seronei Chelulei Cheison Thank you for this reminder. Super helpful

Other answers are also great!