Red blood cell (RBC) membranes are rich in a glycoconjugate that is extractable in chloroform/methanol solutions (2/1, v/v) and contains several hexoses, such as glucose. Old and young RBC are separated and their respective glycoconjugates are prepared. HbA0 is purified by column chromatography and incubated with solutions of this conjugate. After 24-h incubation, Hb is dialyzed and the amount of glycosylated Hb is measured by a method of column chromatography adapted from Trivelli. A very significant amount of HBA1c is formed when young RBC extracts are incubated: 3.6﹪ of total Hb becomes HBA1c with the extracts, versus 3.2﹪ with free glucose, and only 2.5﹪ for controls. No increase in HbA1c is obtained when extracts of old RBC are incubated. Another difference between the action of the glycoconjugate and free glucose is that the former induces the increase of only the HBA1c fraction, whereas glucose induces the increase of all the minor Hb fractions. The evaluation of glucose contained in the conjugate before and after the glycosylation reaction demonstrates that it is due to an exchange of glucose units from the conjugate to Hb. The reaction is stereospecifically inhibited by p-nitrophenyl-β-D-glucoside. The nature of the formed HbA1c is demonstrated by isoelectric focusing. A slight increase of HbA1c observed in the incubated controls may be due to an internal migration of some residues of glucose primitively bound to lysyl residues in an unstable form and also to some degree of denaturation during the incubation.

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