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....
