Sites of nonenzymatic glycosylation of human hemoglobin A

R. Shapiro, M. J. McManus, C. Zalut, H. F. Bunn

Research output: Contribution to journalArticlepeer-review

311 Scopus citations

Abstract

Glucose reacts nonenzymatically with α- and ε-amino groups in the major human hemoglobin component, Hb A(O), to form amino-1-deoxyfructose adducts both in vivo and in vitro. Hb A(Ic), the adduct formed with the NH2 terminus of the β chains, can be isolated by chromatography and comprises about 4% of the total hemoglobin in normal red blood cells. An additional 8 to 10% of Hb A(O) is glycosylated at the NH2 terminus of the α chains or at lysine amino groups. These glycosylated species have not previously been separated from the nonglycosylated Hb A(O). We have examined the specificity of this glycosylation and determined which amino groups are modified in vitro and in vivo. Purified Hb A(O) was incubated with [14C]glucose. Glycosylated peptides were isolated from tryptic digests of this hemoglobin by a combination of ion exchange chromatography and two-dimensional peptide mapping. Amino acid analysis of these peptides revealed the major sites of in vitro glycosylation (in order of prevalence) to be β-Val-1, α-Lys-16, β-Lys-66, β-Lys-17, α-Val-1, α-Lys-7, and β-Lys-120. Identification of sites in native, unincubated Hb A(O) required the development of a method for separating nonglycosylated and glycosylated hemoglobins. This was achieved through anion exchange chromatography by using borate buffers, which form anionic complexes with amino-1-deoxyfructose residues. The glycosylated hemoglobin fraction was labeled with [3H]borohydride and then analyzed in the same manner as the in vitro material. The major sites of glycosylation in order of prevalence were β-Val-1, β-Lys-66, α-Lys-61, β-Lys-17, and α-Val-1. Thus, there are significant differences between the in vivo and in vitro sites of glycosylation.

Original languageEnglish
Pages (from-to)3120-3127
Number of pages8
JournalJournal of Biological Chemistry
Volume255
Issue number7
StatePublished - 1980
Externally publishedYes

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