Sequence of oxygen binding by hemoglobin

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Abstract
A nitroxide spin-label probe was attached directly to a propionic acid group of heme in either the .alpha. or the .beta. chain of Hb. The EPR spectrum of the spin label was altered by the spin-state change of the heme Fe to which the spin label is attached. These hybrid Hb showed normal optical and functional properties, indicating that the attachment of the spin label did not perturb the function of Hb. Upon deoxygenation of .alpha.-heme-spin-labeled Hb, EPR signals changed proportionally with oxygen saturation (determined by measuring absorption spectra). Apparently there is no binding preference between the .alpha. and .beta. chains of Hb. 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. In the presence of organic phosphate, oxygen apparently bound preferentially to the .alpha.-subunit of deoxyhemoglobin. This conclusion was supported by the result obtained with .beta.-heme-spin-labeled Hb: the direction of the deviation for .beta.-heme-spin-labeled Hb in the presence of diphosphoglycerate and inositol hexaphosphate was opposite to that obtained for .alpha.-heme-spin-labeled Hb. The curve deviated even in the absence of organic phosphate. This deviation for .beta.-heme-spin-labeled Hb can be explained by the intersubunit interaction of Hb. 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 Hb.