Re: cross links from diimide with protein

benoiton@aix1.uottawa.ca
Wed, 17 Sep 1997 09:40:48 -0400 (EDT)


Herewith some thoughts on your submission:
1. Carbodiimide is used to make activated esters, so it links COOH with
HOPheX. Your tyrosyl side-chains are phenols, albeit weak, and are
therefore your most likely candidate for cross linking with carboxy
groups.
2. It is not outside the realm of possibility that two tyrosyl side-chains
could end up linked together also, as the phenolic HO could react with
carbodiimide to give the O-alkylisourea, which could react with another
phenolic group.
3. The next candidates, though less likely, would be the the OH of serine.
4. Acylation of the imidazole of histidine could also occur, but the
acyl-imidazole formed would itself be a good acylating agent, so the
product is unlikely to be very stable.
5. Diimides give symmetrical anhydrides, so two COOH groups can also
react together. Again, the products would be 'moderately' stable only.

This probably gives you more possibilities than you hoped for!

Leo Benoiton
benoiton@aix1.uottawa.ca

On Fri, 12 Sep 1997 RLNiece@AOL.com wrote:
~~e of our user groups brought us a sample of carbodiimide cross-linked
roteins (A and B, both sequences known) that had then been subsequently

> One of our user groups brought us a sample of carbodiimide cross-linked
> proteins (A and B, both sequences known) that had then been subsequently
> reduced, alkylated and digested with trypsin. The mass of one of the HPLC
> peaks indicated it could contain cross-linked peptides from proteins A and B
> so we chose to sequence this fraction. In each cycle we saw residues that
> fit a peptide from protein A and residues that fit a peptide from protein B.
> That was nice. However, while both peptides had several acidic residues,
> neither peptide had an internal lysine. Peptide (25 residues long) from A
> also had histidine, methionine, tyrosine, serine and threonine; peptide (70
> residues long) from B had cysteine, tryptophan, histidine, methionine,
> tyrosine, serine and threonine. Of course, both had amino acids like
> glycine, alanine and amides. The question is what other reactions might
> occur in the presence of carbodiimide that cross-links these two peptides.
> It will take more than the 5 pMol of this product that we have to find the
> site of cross-linking. Thanks for considering this problem.
>
>