Aspirin acetylation of betaLys-82 of human hemoglobin. NMR study of acetylated hemoglobin Tsurumai.
Acetylation of hemoglobin by aspirin and other compounds has been of interest for the development of agents useful for the treatment of sickle cell disease. In the present study, we have used 2D NMR methods in combination with [1-(13)C-acetyl]salicylic acid to probe the acetylation sites of hemoglobin A and hemoglobin Tsurumai, a mutant human hemoglobin characterized by a betaLys-82-Gln substitution. In contrast to earlier studies by Klotz and coworkers (e.g. Shamsuddin M, Mason RG, Ritchey JM, Honig GR and Klotz KM, Proc Natl Acad Sci USA 71: 4693-4697, 1974) in which it was concluded that betaLys-144 is the principal target residue acetylated by aspirin, the present study confirms our previous but less conclusive demonstration (Xu ASL, Macdonald JM, Labotka RJ and London RE, Biochim Biophys Acta 1432: 333-349, 1999) that betaLys-82 is the primary acetylation site of aspirin and related agents. The present studies also provide conclusive evidence that acetylation of betaLys-82 produces multiple resonances, probably as a consequence of additional acetylation of other sites, particularly betaLys-82' on the second beta chain. The present results also resolve the apparent discrepancy between the targets of modification by aspirin and double-headed aspirin analogs, and provide an explanation for the changes in oxygen affinity and aggregation threshold of aspirin-modified hemoglobin previously observed under in vitro conditions. In light of the present identification of the principal site of acetylation, the potential therapeutic benefit of aspirin in the treatment of sickle cell disease is discussed.
Xu
A S
AS
Laboratory of Structural Biology, NIEHS, Research Triangle Park, NC 27709, USA.
Ohba
Y
Y
Vida
L
L
Labotka
R J
RJ
London
R E
RE
D000107:Acetylation / Q000187:drug effects; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism / Q000494:pharmacology; D001241:Aspirin / Q000378:metabolism / Q000494:pharmacology*; D006454:Hemoglobins / Q000378:metabolism*; D006455:Hemoglobins, Abnormal / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular
D000894:Anti-Inflammatory Agents, Non-Steroidal; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; C102340:hemoglobin Tsurumai; D008239:Lysine; D001241:Aspirin