EPR Spectral Changes of Nitrosil Hemes and Their Relation to the Hemoglobin T-R Transition

摘要

EPR spectra of nitrosil-hemes were used to study the quaternary structure of hemoglobin. Human adult hemoglobin has been titrated with nitric oxide at pH 7.0 and 25 exp 0 C. After the equilibration of NO among the alpha and beta subunits the samples were frozen for EPR measurements. The spectra were fitted by linear combinations of three standard signals: the first arising from NO - beta hemes and the other two arising from NO - alpha hemes of molecules in the high and low affinity conformations. The fractional amounts of alpha subunits exhibiting the high affinity spectrum fitted the two-state model with L = 7 x 10 exp 6 , and csup( alpha ) sub(NO) and csup( beta ) sub(NO) approximately 0.01. Hemoglobin has been marked with nitric oxide at one chain using low-saturation amounts of nitric oxide. The EPR spectra were studied as a function of oxygen saturation. Linear combinations of the three standard signals above fitted these spectra. The fractions of molecules exhibiting the high affinity spectrum fitted the two-state model with L = 7 x 10 exp 6 , csub(O sub 2 ) = 0.0033 and csup( alpha ) sub(NO) = 0.08, instead of csup( alpha ) sub(NO) = 0.01.Thus, the two state model is not adequate to describe the conformational transition of these hybrids. The results are evidence of the nonequivalence between oxygen and nitric oxide as ligands. (Atomindex citation 13:645104)