Trypsin

YYC szcharng at peseta.ucdavis.edu
Thu Jan 26 21:43:19 EST 1995



On 25 Jan 1995, Patrice Koehl wrote:

> 	Greetings,
> 
> 	My wife has a 54 amino acid long peptide containing 12 cysteines.
> Fully oxydised, the 12 cysteines are involved in 6 disulphide bridges. Though
> the peptide contains 4 sites for trypsin, it does not cut it (which is not
> too surprising, considering the stability the S-S bridges provide)
> After full reduction, and alkylation of all free cysteines with vynil pyridine
> (confirmed by MS), trypsin still does not cut the peptide. This is a little bit
> more surprising, since the S-S bridge cannot form anymore. One hypothesis could
> be that the vynil pyridine help forming a very stable hydrophobic core.
> She has tried to add urea in order to unfold the peptide such that accessibility
> for trypsin is easier, however it did not help. (Urea inhibits trypsin ?)
> Any idea/references on how to "help" trypsin to cut this reduced, alkylated form ?
> Thanks in advance for your help
> 
> Patrice
> 
> Patrice Koehl
> UPR 9003 du CNRS, ESBS			Tel (33) 88 65 53 89
> Boulevard Sebastien Brant		Fax (33) 88 65 53 43
> Illkirch Graffenstaden, France		e-mail : koehl at bali.u-strasbg.fr
> 
> 

I wonder if she can denature the peptide by boiling and then treat 
it with trypsin.  It has been successful for tryptic digestion of a 400-AA 
protein.  Not sure for short peptide.
Yee-yung Charng



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