The principal site of nonenzymatic glycosylation of human serum albumin in vivo

• Published in: The Journal of biological chemistry Vol. 258; no. 10; pp. 6142 - 6146
• Main Authors: Garlick, R L, Mazer, J S
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.05.1983; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acids - analysis; Borohydrides; Chromatography, Affinity; Chromatography, Gel; Glucose - metabolism; Humans; Peptide Fragments - isolation & purification; Serum Albumin - analysis; Serum Albumin - metabolism; Trypsin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)32384-6

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.