Protein Degradation Problem

Eric E. Snyder eesnyder at beagle
Wed Mar 23 16:57:46 EST 1994

I am posting this for a friend w/o usenet access.  Please 
send follow-ups to this address:


We have a problem with a protein that may be helped by someone on the net.
Any remarks would be appreciated.

There is a very weird degradation  of this protein.

N-term   MRPSG....
c-term   ....FQEDE

The protein is stored in citrate at pH 5-6 Several breakdown products have 
been isolated.
1) Modified protein product 1
	electrospray mass spec shows loss of 18 amu from protein

	peptide mapping followed by tandem mass spec showed the 18 amu within
		the C-terminal tryptic peptide and the final two -DE residues

	protein sequencing by Edman degrad. sequencing from N-term of C-term 
		peptide cannot sequence through the penultimate D residue

Oner hypothesis is that the carboxylic acid groups from the D and E residues 
are condensing to form an acid anhydride .  The dehydration product appears to 
be stable in aqueous buffers at 5-6 pH.

2) Modified protein 2

This may be from the specie above or from the parent

	cyclized version of the parent protein in which the C and N termini 
react with the loss of the c-term E residue and formation of a peptide bond 
between the N-term residue and the penultimate Asp residue.

	X-ray crystal structue of the protein show that the N and C are in 
close proximity 
Has any one seen such modifications?
Is there an alternate hypothesis to fit the loss of the 18 amu in the last two 
(DE) residues?
Is there another mechanism for cyclization of the protein through the reaction 
of the N and C termini that would result in the elimination of the c-term  -E

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