Glycosylation of human albumin in diabetes mellitus II. Extensive in vitro modification by trioses and hexoses as revealed by isoelectric focusing

Purified human serum albumin (HSA) from normal adults has been reacted in vitro with different trioses and hexoses and the reaction products analysed by ultrathinlayer isoelectric focusing (IEF) under native and denaturing (8 M urea) conditions. The order of reactivity of these molecules has been found to be: glyceraldehyde > glyceraldehyde 3‐phosphate > glucosamine ≫ glucose ≫ acetaldehyde. By chemical analysis, the number of residues of glyceraldehyde bound to albumin appears high: 130 moles/mole of protein, while the number of exposed lysines is halved: ca. 8 in reacted vs. 16 in control albumin. It appears that a minimum length for an aldehyde to react with protein amino groups is three carbon atoms. A completely different IEF behavior has been found between “reacted and NaCNBH 4 reduced” vs. “reacted and rearranged” HSA molecules. The former group, even when extensively reacted with sugars, exhibits a normal surface charge at pH = p I , possibly because, at the acidic p I s of HSAs, the now secondary (sugar‐bound) amino groups are still fully protonated. In the latter group, the bound sugar spontaneously undergoes an Amadori rearrangement followed by secondary reactions, still not fully clarified, which could possibly lead to ring structures ( e. g. pyrrolic) with complete loss of positive charge from the sugar‐reacted amino groups. These species, in fact, show a markedly more acidic p I .