Monosaccharide Analysis

Robert B. Trimble trimble at news.wadsworth.org
Mon Apr 24 14:57:08 EST 1995


Dear Pat;

We have done monosaccharide analysis of glycoprotein oligosaccharides for
many years using a 2N HCl/100C/2 h protocal, which gives excellent results
for neutral sugars. Aminosugars are analyzed after 6N HCl/100c/12 h. We use
rhamnose or fucose as an internal standard for neutral sugars, and GalN when
assaying GlcN. Our experience more recently with TFA hydrolysis has given
the same results you report, loss of mannose. We did some time-course studies
using TFA in collaboration with Reid Townsend at UCSF Dept. of Pharm. Chem.,
and saw loss of mannose over time with a comcomitant increase in glucose. 
My suspicion is that, for reasons chemically unknown to me, we're seeing 
some epimerization about the C-2 under the 4N TFA conditions used in the 
literature.

The old 2N HCl method works well if you use a fresh stock of C.B. HCl, 20
megohn water, acid washed hydrolysis tubes, and flush the tubes with pre-
purified nitrogen and seal under vacuum. The HCl will blow off by flash 
evaporation from water in three cycles. Also, we have found that monosacch-
aride analysis of whole glycoproteins especially requires hydrolysis under
reduced pressure after nitrogen flushing. I suspect that in air some of the
amino acids produce oxidation products that interfere with recovery of neutral
hexoses. I thought that others in the newsgroup might find thi comment use-
ful. I've sent a more in-depth reply to your e-mail address.

Rob Trimble
Wadsworth Center C535
NYSDOH, Albany, NY 12201-0509
e-mail: Robert.Trimble at wadsworth.org



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