Single peaptide is 2 peaks on RP-HPLC. Why?

Mr. P.A. Sansom psansom at hgmp.mrc.ac.uk
Sat Jan 14 15:21:35 EST 1995


Single peptide is 2 peaks on RP-HPLC. Why?

Background
I'm using C18 reverse phase HPLC (TFA and acetonitrile gradients) to 
separate fragments of a digested peptide. The problem is that the starting 
peptide (16 amino acids, sequence NH2-DHLSDNYTLDHDRAIH-COO) detected at 
206nm runs as two equal height overlapping peaks, not separable. It is 
synthetic, but sequence analysis and mass analysis show it to be pure, ie 
there is only one main species present. Also, I've had three separate 
preparations made at two different sites, and the effect is seen in all of 
them!

Trypsin treatment cleaves AIH from the C-terminal resulting in a single peak
corresponding to DHLSDNYTLDHDR and a small double peak corresponding to 
AIH indicating that the variation lies in this AIH region.

The only suggestions I've had are that his-16 may be breaking open, or that
it's a conformational effect caused by an inflexible ile-his bond.

If anyone can confirm, deny, or offer an alternative I'd be grateful.

Thanks in advance

Paul

psansom at hgmp.mrc.ac.uk

Paul A Sansom                                  tel: 081-748-9966 x4208
Department of Biochemistry                     fax: 081-748-5090
The Kennedy Institute of Rheumatology
6 Bute Gardens
Hammersmith
London W6 7DW



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