Sequence of oxygen binding by hemoglobin.
A nitroxide spin-label probe was attached directly to a propionic acid group of heme in either the alpha or the beta chain of hemoglobin. The electron paramagnetic resonance (EPR) spectrum of the spin label is altered by the spin-state change of the heme iron to which the spin label is attached. These hybrid hemoglobins showed normal optical and functional properties, indicating that the attachment of the spin label did not perturb the function of hemoglobin. Upon deoxygenation of alpha-heme-spin-labeled hemoglobin, EPR signals changed proportionally with oxygen saturation (determined by measuring absorption spectra). This result indicates that there is no binding preference between the alpha and beta chains of hemoglobin. However, the cross plot for the fraction of the EPR changes vs. the fraction of oxygen saturation deviated significantly from the diagonal straight line in response to the addition of 2,3-diphosphoglycerate and inositol hexaphosphate. The deviation indicated that the EPR change precedes the optical change at low oxygen tension. This result implies that, in the presence of organic phosphate, oxygen binds preferentially to the alpha subunit of deoxyhemoglobin. This conclusion was supported by the result obtained with beta-heme-spin-labeled hemoglobin: the direction of the deviation for beta-heme-spin-labeled hemoglobin in the presence of diphosphoglycerate and inositol hexaphosphate was opposite to that obtained for alpha-heme-spin-labeled hemoglobin. However, the curve deviated even in the absence of organic phosphate. This deviation for beta-heme-spin-labeled hemoglobin can be explained by the intersubunit interaction of hemoglobin. From these results, it was concluded that, in the absence of organic phosphate, oxygen combines with the alpha and beta chains with equal probability whereas, in the presence of organic phosphate, oxygen binds preferentially to the alpha chains of hemoglobin.
D000328:Adult; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme; D006454:Hemoglobins; D006801:Humans; D007700:Kinetics; D010100:Oxygen / Q000097:blood; D010108:Oxyhemoglobins; D011485:Protein Binding; D013113:Spin Labels
D006454:Hemoglobins; D010108:Oxyhemoglobins; D013113:Spin Labels; D006418:Heme; D010100:Oxygen