NMR study of the sites of human hemoglobin acetylated by aspirin.
Acetylation of hemoglobin by aspirin and other acetylating agents has been used to generate hemoglobin analogs with altered structural and functional properties, and may prove useful in the treatment of sickle cell disease. We have studied the acetylation of human hemoglobin using [1'-(13)C]acetylsalicylic acid in combination with two-dimensional HMQC and HSQC NMR analysis. The spectra of the acetylated hemoglobin exhibit a number of well resolved resonances. Several spectral assignment strategies were used: blocking the 2, 3-DPG binding site non-covalently with inositol hexaphosphate or covalently with a cross-linking agent, selective carbamylation of the N-terminal valine amino groups with cyanate, spin-labeling the hemoglobin at betaCys93, and analysis of a hemoglobin triple mutant: betaV1MH2DeltaK144R, in which betaLys144 is replaced by an arginine residue. These studies support the conclusion that the most rapidly acetylated residue is betaLys82 rather than betaLys144, as previously reported. Further, it is apparent that acetyl betaLys82 can give rise to several resonances due to additional acetylation of betaLys82' or other nearby residues. An additional assignment strategy involving comparison of the chemical shifts of the acetyl resonances observed for adducts of diamagnetic carbonmonoxyhemoglobin with the shifts observed in paramagnetic cyanomethemoglobin provides information about the location of the acetyl derivatives relative to the heme irons. This approach is limited, however, by the lack of well defined structural information for the lysine residues on the protein surface. Additional tentative assignments have also been made, using the above approaches.
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709-2233, USA.
A S
AS
Xu
J M
JM
Macdonald
R J
RJ
Labotka
R E
RE
London
D019794:2,3-Diphosphoglycerate / Q000737:chemistry; D001241:Aspirin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry*; D001665:Binding Sites; D002247:Carbon Isotopes; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008706:Methemoglobin / Q000031:analogs & derivatives / Q000737:chemistry; D009154:Mutation; D010833:Phytic Acid / Q000737:chemistry; D013113:Spin Labels
D002247:Carbon Isotopes; D006454:Hemoglobins; D013113:Spin Labels; C058244:acetylated hemoglobin; C007169:cyanomethemoglobin; C050588:bis(2,3-dibromosalicyl)fumarate; D019794:2,3-Diphosphoglycerate; D010833:Phytic Acid; D008706:Methemoglobin; D001241:Aspirin