To determine the mechanism for the formation of hemoglobin Alc (Hb Ale) in vivo, we incubated human hemoglobin with glucose and metabolites of glucose. ['4CJGlu-cose-6-phosphate (G6P) reacted readily with deoxyhemoglobin, and formed a covalent linkage. The reaction rate was considerably reduced in the presence of carbon monoxide or 2,3-diphosphoglycerate (2,. Purified G6P hemoglobin had a lowered oxygen affinity and decreased reactivity with 2,3-DPG compared to Hb A. G6P behaved as a 2,3-DPG analog and reacted specifically at the NHrterminal amino group of the P chain. In contrast, the interaction of hemoglobin with glucose was much slower, and was unaffected by carbon monoxide or 2,3-DPG. Neither glucose-i-phosphate, fructose-1-phosphate, fructose-6phosphate, nor fructose-1,6-diphosphate formed a reaction product with hemoglobin. G6P action. We have examined the interaction of human hemoglobin with glucose and glucose metabolites in a cell free system devoid of enzymes or cofactors. Our results indicate that 1)-glucose-6-phosphate (G6P) readily forms a covalent linkage specifically at the NH2-terminus of the fl chain, while glucose does not.
MATERIALS AND METHODSBlood from normal adults was collected in heparin or EDTA. Hemolysate was prepared by the method of Drabkin (8). Hb A was purified at 40 on a carboxymethylcellulose column (CM 52, Whatman, Inc.; Clifton, N.J.) (9). Approximately 2 g of hemoglobin was applied to a 4.5 X 25 cm column, and eluted for 15 h with 0.01 M phosphate buffer containing a linear pH gradient (pH 6.8-9.0) at a rate of 90 ml/hr.Kinetics of Hemoglobin Modification. Hemolysate was placed in two-dimensionally stretched cellophane tubing and dialyzed exhaustively against 0.1 M Tris-HCl at pH 7.2. Two milliliters of the stripped (phosphate-free) hemoglobin was placed in a gas-tight vial and either deoxygenated by passing hydrated argon across the solution, or saturated with carbon monoxide (CO).[14C]G6P (New England Nuclear, Boston, Mass.) (50,MCi, 0.95 jimol) was mixed with 35.5 Mtmol of unlabeled G6P in 0.5 ml of distilled water and deoxygenated in a similar manner. In like manner, 50 uCi (10.5 umol) of D-[14C]glucose was mixed with 26.1 umol of unlabeled 1-glucose in 0.5 ml of distilled water and deoxygenated. The deoxy Hb solution was then mixed with the deoxygenated glucose or G6P solution. In a parallel experiment, we tested the interaction of purified hemoglobin A with unlabeled glucose-l-phosphate, fructose-l-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and glucuronic acid (Sigma Chemical Co.