Glycosylated Minor Components of Human Adult Hemoglobin

Human hemolysate contains several minor components designated Hb A,,, Hb Alh, Hb A,,, which are post-translational modifications of the major hemoglobin component A,,. Individuals with diabetes mellitus have elevated levels of Hb A,,, a hemoglobin modified with a glucose moiety at the NH, terminus of each /3 chain. A new chromatographic technique using Bio-Rex 70 is described which not only allows complete separation of Hb A,, from Hb A,, but also resolution of Hb A,, into two components, designated Hb A,,, and Hb A,,,. Carbohydrate determinations with the thiobarbituric acid procedure revealed that Hb Ala1, Hb A laq, and Hb A,, as well as Hb A,, were glycosylated. Total phosphate analysis revealed 2.06 and 1.01 mol of phosphorus/a/3 dimer for Hb A,,, and Hb A,,, respectively; Hb A,, and Hb A,, contained no detectable phosphate. Hemoglobin incubated with o-[14C]ghtcose-6-P co-chromatographs precisely with Hb A,,,, strongly suggesting that Hb Ala:, is glucose-6-P hemoglobin. Levels of Hb AlaI and Hb A,,, are normal in individuals with diabetes mellitus. Furthermore, diabetic red cells contain normal levels of glucose-6-P. Therefore, glucose-6-P hemoglobin does not serve as a . . . significant precursor to Hb A,,. Instead Hb A,, is formed by the direct reaction of hemoglobin with glucose. This suggests that hemoglobin can serve as a model system for nonenzymatic glycosylation of protein.