Re: tryptic digest
Rod Levine (rlevine@nih.gov)
Thu, 26 Feb 1998 07:49:16 -0500
At 09:49 AM 2/24/1998 -0800, Deb McMillen wrote:
>Hi, all,
>I have a client who is getting cleavage of a protein between D and P with
>trypsin (Sigma Type 13 TPCK treated bovine pancreatic). When he starts
>the cleavage, the protein is intact. Of course, the first thing that
>comes to mind is how labile the DP bond is, but doesn't it take at least
>acidic conditions perhaps with elevated temperature to break the bond?
>The tryptic digestion (partial) is done at room temperature in 10 mM
>Hepes, 150 mM NaCl, 0.1 mM EDTA, 0.5mM DTT, 10% glycerol pH 7.8. They are
>using 50 micrograms of trypsin per 500 micrograms of protein. Is
>one of the reaction mixture components a strong (or weak) enough acid to
>attack the DP bond?
Deb,
As you and other responders noted, we usually think of acid-catalyzed
cleavage of the Asp-Pro bond. However, I have at least 2 references which
suggest that it can occur at higher pH, specifically in "SDS buffer". One
of them shows that even a minute or so at 100 C can cause significant
cleavage. I've always assumed that if this occured for most proteins, then
there could be some where this occured at room temperature, presumably
related to the particular sequence.
Anyway, you might find these references interesting:
Anal Biochem 123:86 1982
Anal Biochem 138:442 1984
Rod Levine
NIH
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