H-1-NMR STUDY ON GANGLIOSIDE AMIDE PROTONS - EVIDENCE THAT THE DEUTERIUM-EXCHANGE KINETICS ARE AFFECTED BY THE PREPARATION OF SAMPLES

The kinetics of H/H-2 chemical exchange of the amide proton has been suggested as one of the tools available for investigating hydrogenbond stabilizing interactions in gangliosides. The amide proton/deuterium (NH/H-2) exchange rates in GM2 ganglioside were studied by H-1-NMR spectroscopy on 12 samples prepared following different procedures. In samples passed through a sodium salt Chelex-100 cation exchange resin column prior to being analysed the N-acetylneuraminic acid NH exchange occurred in less than 10 min and that of ceramide NH in 30 min. The N-acetylgalactosamine acetamido NH exchange was slower, the half-life of the signal ranging from 15 min to 3.5 h. Contact of the Chelex-treated GM2 samples with water, through a dialysis process, modified the NH/H-2 exchange rate values, the N-acetylgalactosamine acetamido NH exchange becoming faster than that of ceramide NH and similar to that of N-acetylneuraminic acid NH. Our results indicate that the deuterium/proton exchange rate strongly depends on sample preparation (ion content and minor contaminants present in water). The three-dimensional model involving the N-acetylgalactosamine acetamido NH and the N-acetylneuraminic acid carboxyl group hydrogen-bonding, which is supported by experimental evidence, cannot be confirmed by NH-exchange measurement.