disulphide bridges?????

Anton Scott Goustin asg at cmb.biosci.wayne.edu
Mon Jul 17 16:57:54 EST 1995


On Bastille Day, Mark Pallen <m.pallen at ic.ac.uk> wrote:
>Hi!
>
>A colleague of mine, Gadi Frankel (gfrankel at molbiol.ox.ac.uk) has asked 
>me to post the following request:
>
>If you have a protein with two or more cyteines in it, what is the best 
>and/or easiest biochemical method for determining which, if any, pair 
>or pairs of cysteines is/are linked via a disulphide bridge?
>
>please e-mail as well as post.
>thanks in advance
>
>Mark
>********************************************************
>Dr Mark Pallen, Senior Lecturer in Medical Microbiology,

Mark,
This is not easy.  People have generally taken the approach to isolate 
the non-reduced protein, with exquisite measures to prevent scrambling 
of the -S-S- bonds, including keeping the pH low, etc.  Mike Waterfield 
at the Ludwig Institute in Middlesex has published a method, I believe:
    http://www.ludwig.ucl.ac.uk/
In general, you subject the purified protein to tryptic digestion, for 
example, and purify the chief tryptic fragments using HPLC.  Identify 
the peaks containing CYS.  Subject those peaks (if pure) to 
microsequencing.  If the peak contains a pair of tryptic peptides linked 
by -S-S- bonds, the sequence will reveal (usually) two different aa at 
each cycle, corresponding to the sequence of each primary chain.  I am 
assuming that you have complete (or nearly) peptide sequence information 
on your protein....





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