disulphide location in short peptides

[John A. Newitt]

In article <3uch22$15r at nuscc.nus.sg>, medp4044 at leonis.nus.sg (Gurmil Singh
s/o Dalip Singh) wrote:

> Dear Netters,
> 
> I am currently working on the secondary structure of a short peptide of 
> 35 amino acids. It got 6 Cys and all are involved in disulphide bonds 
> i.e. 3 of them. I am trying to digest my peptide in the un-reduced form 
> in order to determine the location of the disulphides, however, none of 
> the enzymes I have used seems to work. These include Glu-C, Lys-C, 
> Chymotrypsin, Trypsin and Pepsin. Stringent denaturing treatment of the 
> peptide prior to digestion i.e. 8 M Urea or 6 M Guanidine-HCl proof to be 
> ineffective as well. Any suggestions on how I can get around this problem 
> and get my disulphide location will be most welcomed.
> 
I've never done this myself, but this might work.  Denature with 0.5-0.1%
SDS (the higher the SDS the faster the denaturation, but Glu-C loses
significant activity at 0.1% SDS; heat to 65 degrees C for 30 minutes),
dilute with buffer to reduce SDS conc. to 0.05-0.01%, then add Glu-C to
1/20 the weight of peptide substrate.  Digest at 25 degrees C for 2-18
hours.  You could try using either ammonium carbonate pH 7.8 or ammonium
acetate pH 4.0 to cleave at glutamic acid residues, or phosphate pH 7.8 to
cleave at glutamic and aspartic residues.  Of course, all bets are off if
your peptide contains neither of these residues.  Do you have an amino
acid composition of this peptide?

Regards,

John A. Newitt, Ph.D.           |   <newitt at nih.gov>
National Institutes of Health   |   Tel: 301-402-4770
Bethesda, Maryland  USA         |   FAX: 301-402-0387