Chain nonequivalence in binding of nitric oxide to hemoglobin.

In the presence of inositol hexaphosphate (IHP), the ESR spectrum of each of the NO derivatives of the human HbA/cat HbA hybrid hemoglobins, a~“&~ and aZCPZH, resembles the spectrum of the parent hemoglobin that provides the a chain, particularly with respect to the intensity of the triplet hyperfine structure at g = 2.009. This observation gives support to the suggestion (Henry, Y., and Banerjee, R. (1973) J. Mol. Sol. 73, 469-482; Peru@ M. F., Kilmartin, J. W., Nagai, K., Szabo, A., and Simon, S. F. (1976) Biochemistry 15, 378-387) that the hyperfine structure is an a chain specific spin label within T state NO-hemoglobin. The question of chain nonequivalence in the equilibrium binding of NO to T state hemoglobin was investigated using relative ESR resonance intensities at g = 2.009 and g = 1.985 to estimate the fraction of NO bound to the a-hemes. Partially saturated solutions of nitric oxide hemoglobin prepared by mixing solutions of NO and deoxyhemoglobin with IHP or of NO-hemoglobin and deoxyhemoglobin with IHP were allowed to equilibrate at room temperature prior to analysis by ESR spectroscopy at 77 K. The hyperfine structure at g = 2.009 was much more prominent in the spectra of solutions at low than at high degrees of saturation, indicating that a greater fraction of the NO was bound to the a than to the fi chains at low saturation. A timedependent increase in the ESR hypefine structure intensity occurs when a solution of saturated NO-hemoglobin is mixed with a solution of deoxyhemoglobin plus IHP and is allowed to equilibrate. This suggests that NO dissociates and then recombines preferentially with available a chain heme sites in T state hemoglobin. Half-times for attainment of equilibrium under the conditions of these experiments were of the order of 5 min at room temperature.