The Case of the Missing Residue
Hi,
I wonder if anybody can help shed any light on a problem I've been having
recently.
I'm isolating some chondroitin sulphate oligosacharides by digestion with
Chondroitinase. The action of this enzyme results in an unsaturated C=C in
the non-reducing terminal residue. Now if you have ever worked with
chondroitin sulphate oligosaccharides you will know that this bond has
some dramatic features - it absorbs at 232nm and has very distinctive
proton NMR signals.
So, I depolymerise my chondroitin sulphate chain and isolate the resulting
oligosaccharides by ion exchange chromatography. I've got my oligos but
they are in the salt used to elute them (Lithium Perchlorate) so I desalt.
Everything is fine - I've got my nice pure oligos - but in a couple of cases
I've had this terminal residue with the unsaturated bond disappear!
Now what do I mean by disappear - well
The (proton) NMR spectra have altered - the signals characteristic of this
bond have gone (I know they were there because I've acquired a spectrum
but now they are gone) - however I cannot at this time locate any signals
which have appeared in the place of those which are gone.
The absorbance of the oligo at 232nm has fallen (this wavelength is used to
monitor the products of chondroitinase digestions as they absorb strongly
at 232nm).
It has always happened in an NMR tube in D2O, plus phosphate buffer and
TSP (reference). In one case it happened within four weeks (I've had other
samples in tubes for months with no ill-effects)
So, the main question is has anybody even seen this phenomena before?
I need to examine the spectra more closely to see if I can figure out what
has happened and how it has come about.
But, like I say if anybody has seen this before I'd appreciate your thoughts
Bob Lauder
R.Lauder at Lancaster.ac.uk
