RE: tryptic digest

Amina (amina@welchlink.welch.jhu.edu)
Thu, 19 Nov 1998 11:05:09 -0500

I routinely see Lys/Arg cleavage with trypsin, using Boehringer sequencing
grade Trypsin, diluted in 0.001N HCl using enzyme:substrate ratios of 1:10
or 1:20, and 25mM Ammonium Bicarbonate pH 8.5 for buffer. I would have to
go back and look if the presence of Gly makes a difference. I know that
with Endoproteinase Glu-C (Boehringer sequencing grade) Glu residues
preceded by Ala or Gly get cleaved (at a much slower rate) on their amino
terminal end as well as their carboxy terminal conventional cleavage site.
I'm wondering if It's possible that these two residues tertiary structures
give room to these enzymes to perform cleavages they would not be able to
do otherwise.

Amina

Amina S. Woods, Ph.D.
Johns Hopkins School of Medicine
725 N Wolfe St., Baltimore, MD 21205
Tel: (410) 614-4981, Fax: (410) 955-3420
E-mail: amina@welchlink.welch.jhu.edu

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From: Peter Hunziker [SMTP:phunzi@bioc.unizh.ch]
Sent: Thursday, November 19, 1998 2:37 AM
To: Recipients of ABRF List
Subject: tryptic digest

long time ago my supervisors teached me that trypsin does never cleave the
peptide-bond between LYS/ARG and PRO. Now, I also tell my students that
this bond is not cleaved at all. However, in a recent LC/MS analysis we
measured the masses of two peptides that indicated a tryptictic cleavage
exactly at this bond (Enzyme: mod. trypsin from Promega). Since I could not
believe it we additionally sequenced the peptides by Edman degradation and
it turned out that trypsin has cleaved the LYS-PRO bond.
On the C-terminal side of the cleavage site there were a number of GLY
residues. Does anybody know if this may influences the specificity of the
enzyme? Were my supervisors wrong and trypsin does cleave LYS/ARG-PRO
bonds?

best regards
Petre

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