J Biol Chem, Vol. 274, Issue 38, 26629-26632, September 17, 1999

COMMUNICATION
Acetylation of Human Hemoglobin by Methyl Acetylphosphate
EVIDENCE OF BROAD REGIO-SELECTIVITY REVEALED BY NMR STUDIES

Arron S. L. Xu, Richard J. Labotka^§, and Robert E. London

From the  Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709-2233 and the ^§ Department of Pediatrics, University of Illinois, Chicago, Illinois 60612-7324

The development of chemical modification agents that reduce the tendency of sickle hemoglobin (HbS) to aggregate represents an important chemotherapeutic goal. Methyl acetylphosphate (MAP) has been reported to bind to the 2,3-diphosphoglycerate (2,3-DPG) binding site of hemoglobin, where it selectively acetylates residues, resulting in increased solubility of HbS. We have prepared [1-¹³C]MAP and evaluated the adduct formation with hemoglobin using ¹H-¹³C HMQC and HSQC NMR studies. These spectra of the acetylated hemoglobin adducts showed 10-11 well resolved adduct peaks, indicating that the acetylation was not highly residue specific. The chemical shift pattern observed is in general similar to that obtained recently using [1'-¹³C]aspirin as the acetylating agent (Xu, A. S. L., Macdonald, J. M., Labotka, R. J., and London, R. E. (1999) Biochim. Biophys. Acta 1432, 333-349). Blocking the 2,3-DPG binding site with inositol hexaphosphate (IHP) resulted in a selective reduction in intensity of adduct resonances, presumably corresponding to residues located in the 2,3-DPG binding cleft. The pattern of residue protection appeared to be identical to that observed in our previous study using IHP and labeled aspirin. Pre-acetylation of hemoglobin using unlabeled MAP, followed by acetylation with [1'-¹³C]aspirin indicated a general protective effect, with the greatest reduction of intensity for resonances corresponding to acetylated residues in the 2,3-DPG binding site. These studies indicated that both MAP and aspirin exhibit similar, although not identical, acetylation profiles and target primarily the Lys-82 residue in the 2,3-DPG binding site, as well as sites such as Lys-59 and Lys-90, which are not located in the -cleft of hemoglobin.