BNPS-Skatole protocol/Side-reactions of CNBr cleavage
Keith Hoek -BIOCHEM
hoek at biosci.uq.oz.au
Thu Sep 21 17:12:31 EST 1995
About your question on the net (20/9/95)
I can't tell you much about BNPS-Skatole, except that I seem to remember
it was a real bugger to put into solution. However, I've played with
CNBr a bit, so I might be able to help you out.
I once cleaved a 26-mer containing one methionine with CNBr as according
to Allen (1989), I then analysed the reaction mix with mass spectrometry.
I got three separate species showing up, plus a very small (and yet
persistent fourth) signal. I had expected a fragment of 1448 Da and
another of 1718 Da, in addition to these I got a lot of 1746 Da and
a little 1774 Da. To cut a long story short, it turned out that the
formic acid in my reaction was formylating the one serine residue in a
significant fraction of the 1718 Da fragments (adding 28 Da to the mass)
and in a smaller fraction both the serine and a single threonine were
being formylated (adding 56 Da to the mass). To remedy this I replaced
formic acid with acetic acid, and the reaction worked fine (I only saw
the expected 1448 and 1718 Da species). There are complications involved
with cleaving Met-Ser and Met-Thr bonds, and these are described in
Allan (1989) and Schroeder et al. (1969).
The formylation stuff is confirmed in another paper (Goodlett et al.,
1990) that you may want to check out.
Allen, G. 1989. In: Laboratory techniques in biochemistry and molecular
biology (Burdon, R.H. and Van Knippenberg, P.H., eds.), 2nd edition,
vol 9, pp. 95-99. Elsevier, Amsterdam, New York.
Goodlett, D.R., Armstrong, F.B., Creech, R.J. and Van Breemen, R.B. 1990.
Formylated peptides from cyanogen bromide digests identified by fast atom
bombardment mass spectrometry. Anal. Biochem. 186, 116-120.
Schroeder, W.A., Shelton, J.B. and Shelton, J.R. 1969. An examination
of conditions for the cleavage of polypeptide chains with cyanogen
bromide: Application to catalase. Arch. Biochem. Biophys. 130, 551-556.
Keith Hoek hoek at biosci.uq.edu.au
intracellular mRNA transport
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