Derek
ext 5442
-----Original Message-----
From: Ellison, Derek
Sent: 23 June 2000 12:21
To: 'Jun Ouyang'
Subject: RE: Chymotrypsin
Chymostrypsin like all proteases shouldn't be thought of solely in terms of
primary sequence specificity - that isn't how they work. There are a large
number of factors that determine where a protease will cleave. The most
important being tertiary/secondary structure. You can demonstrate this in
numerous ways by incubating native proteins with a protease and following
the kinetics of digestion by RP-HPLC, there will always be a preferred first
point of cleavage. The most important factors are the degree of exposure and
flexibility of the loop where the protease is to cleave. Going back to basic
enzymology, the substrate and protease have to be able to 'fit'. If you are
talking about a peptide substrate with no structure, then it's a bit
different. A protease will bind on the basis on a number of sites/amino
acids before and after the cleavage point.
Chymotrypsin is relatively unspecific, an will cut after leucine, but it may
prefer to cleave at other residues in preference first - you may have to
keep the digest going for orders of magnitude longer. It will work fine at
pH 7.5-8.0, use a good buffer (e.g. HEPES, Tris if you work in a constant
temperature environment) and have a few mM CaCl2 in there. (don't use a
buffer that will precipitate calcium like phosphate). Calcium stabilises the
protease and reduce autolysis. It's a poor exopeptidase so if you are using
a peptide substrate and leucine is near the end - it will perform very
slowly.
I could go on about this all day but you've probably heard enough!
Derek
ext 5442
> -----Original Message-----
> From: Jun Ouyang [mailto:Jun.Ouyang.B@bayer.com]
> Sent: 22 June 2000 17:41
> To: Recipients of ABRF List
> Subject: Chymotrypsin
>
>
> Would some experts share experience with me that under what condition
> chymotrypsin will definitely cut after Leucine?
>
> Thanks in advance.
>
> Jun Ouyang
> Bayer Corp.
>
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