-----Original Message-----
From: keehongkim [mailto:khkim@lamar.colostate.edu]
Sent: Wednesday, August 16, 2000 11:15 AM
To: Recipients of ABRF List
Cc: Ken Reardon
Subject: 2nd Best Digestive Enzyme.
<snip>
5. V8 (D,E)
6. V8 (E)
Has anybody REALLY seen a difference between preferred cleavage sites when
using V-8 (Glu-C) in different buffer systems or is this just another
"myth" about this protease? The claim that Glu and Asp are cleaved in
phosphate 7.8 and that Glu only is cleaved in NH4 bicarb 7.8 just hasn't
held up in my experience. Using the SAME mM concentration for each buffer
I've really found about the same amount of cleavage (at D and E) for each
system. In my hands Glutamic acid has always been a much better substrate
for V-8/Glu-C than Aspartic Acid no matter what the buffering system. Asp
cleavages are just a bonus, or an irritation, if you are trying to produce
the perfect protein mapping fingerprint. It seems to be very sequence
dependent. I've tried using Glu-C on at least three different proteins
using the two different buffering systems and have found them to really
produce no difference in cleavage patterns. The area for each peptide was
essentially the same. I've found that if you really need the Asp cuts just
follow the Glu-C two hours later with Asp-N. You will get a "few" ragged
+115/-115 peptides depending on just how active the Glu-C is to that
particular Aspartic acid. Not acceptable for the perfect mapping
fingerprint but just fine for whittling down a peptide to find out the site
of phosphorylation or disulfide bonding patterns.
pH 4.0 digestion with Glu-C? Any takers? So far I haven't heard of one
person that has actually made this enzyme work at low pH. It has been
suggested that this is a different type of enzyme (like the Lys-C enzymes
that are currently available) than was originally produced. If you have
seen digestion at pH 4.0 (other than Asp/Pro) approximately how much was
cleaved?
Gary Lange
gary.w.lange@monsanto.com
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