Spin-labeled hemoglobin.

In the present paper we describe the results of a preliminary study of the paramagnetic resonance spectrum of spin-labeled horse hemoglobin in solution. Our results provide strong evidence that on oxygenation the A chains undergo a significant structural change near the "reactive" sulfhydryl group (f693), a change not yet resolved by X-ray diffraction.' We also find features in the paramagnetic resonance spectra of nitroxide-maleimide spin-labeled hemoglobin that appear to be closely related to observations by Benesch and Benesch2 (BB) on the inhibition of the Bohr effect by N-ethyl maleimide (NEM). Materials and Methods.-Horse hemoglobin was purchased from Calbiochem, 2X crystallized, lot 62248. Fresh hemoglobin was obtained from defibrinated horse blood (W. T. Bennett Ranch Laboratory) according to the method described by Benesch and Benesch.3 Deoxygenation of the hemoglobin was effected by passing high-purity nitrogen over the stirred solution. Since the addition of a small excess of sodium dithionite yielded the same results, this more convenient and more effective method was used preferentially whenever feasible.4 The preparation of the nitroxide-maleimide I (N-( l-oxyl-2,2,5,5-tetramethyl-pyrrolidinyl)-maleimide) is described by Griffith and McConnell.'