The principal site of nonenzymatic glycosylation of human serum albumin in vivo.
We have determined the major site of nonenzymatic glycosylation of human serum albumin in vivo. This was accomplished by reacting freshly purified human serum albumin with sodium [3H]borohydride followed by aminoethylation and tryptic digestion. The tryptic peptides were separated into a soluble fraction which contained 88% of the total 3H radioactivity and an insoluble fraction. In order to isolate the 3H-labeled glycosylated peptides, the soluble tryptic peptide fraction was first subjected to boronic acid affinity chromatography. Cation exchange chromatography then separated the soluble glycosylated peptides into a major peak which contained 48% of the total recovered 3H radioactivity and a number of minor peptide fractions. The amino acid composition of the major peptide was: Thr, Glu2, Ala, Val2, Leu2, Lys, lysino-1-deoxysorbitol. In accord with the primary structure of human serum albumin, this amino acid composition corresponds precisely to residues 525-534. Glucitol-lysine, the NH2-terminal residue of this peptide, is totally resistant to cleavage by trypsin. Thus, lysine-525 is the predominant site of nonenzymatic glycosylation of human serum albumin in vivo. Chromatography on GlycoGel B boronic acid affinity gel indicates that 10-12% of normal serum albumin is glycosylated. The rate of nonenzymatic glycosylation of this protein in vivo is approximately 9 times that of human hemoglobin.
D000596:Amino Acids / Q000032:analysis; D001894:Borohydrides; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005947:Glucose / Q000378:metabolism; D017127:Glycation End Products, Advanced; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification; D012709:Serum Albumin / Q000032:analysis / Q000378:metabolism*; D014357:Trypsin; D000093423:Glycated Serum Albumin
D000596:Amino Acids; D001894:Borohydrides; D017127:Glycation End Products, Advanced; D010446:Peptide Fragments; D012709:Serum Albumin; C025364:sodium borohydride; D014357:Trypsin; D005947:Glucose; D000093423:Glycated Serum Albumin