Hi Ting,
I just came across a paper that talks about the conditions for use of trypsin, including the effect of conc. of denaturant. May it help.
Anal. Biochem. 266, 31-47, 1999
Wei Wu
Department of Chemistry
Michigan State University
East Lansing, MI 48824
1-517-353-6767
----- Original Message -----
From: Rod Levine
To: Recipients of ABRF List
Sent: Friday, August 18, 2000 4:48 PM
Subject: re: trypsin activity in guanidine hydrochloride
At 09:48 AM 8/18/2000 -0600, Ting Wong wrote:
I have encountered incomplete digestion of protein using trypsin. Since
I was given the impression that trpytic digestion of protein can be done
in mild denaturing condition such as 1-2 M 0f guanidine hydrochloride, I
tried that but was not successful. The trypsin seemed to lose activity
even at S/E (w/w) of 5 to 1 (20:1 was used in normal codition). I wonder
if there is anything that I am not aware of in applying such condition
for trypsin. Thanks in advance for your advice.
Ting,
Another myth -- often perpetuated in lists of proteases -- is that trypsin is stable to such concentrations of trypsin. We did a few experiments to assess that point because we had virtually the same experience as you. Those experiments simply confirmed our initial impression -- trypsin doesn't die completely in 1 M guanidine, but it's seriously injured. The rate of peptide bond hydrolysis drops substantially, some bonds which should be cut not act resistant, and cleavage rarely goes to completion.
Rod Levine
NIH
Bldg 3, Room 106 MSC 0320
Bethesda, MD 20892-0320
email: rlevine@nih.gov
voice: 1 (301) 496-2310
fax: 1 (301) 496-0599
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