Non-Equivalence and Inverse Allosteric Response of the α and β Chains in Haemoglobins. An Electron Spin Resonance Study of NO-Ligated Hb Kansas

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Abstract
The ESR spectra of 15NO- and 14NO-ligated Hb Kansas were measured at 77.degree. K in the range of pH 5-10. At low pH, the ESR spectrum is the composite of a type I and a type II spectrum which changes to another composite of a type I and II spectrum at high pH. At intermediate pH values, the ESR spectra of NO-Hb Kansas are the composites of 4 spectral components. The assignments of the 4 spectral components to the .alpha. and the .beta. chains are arrived at from the comparison of the ESR spectra of the .**GRAPHIC**. and of the .**GRAPHIC**. species of Hb M Iwate; .alpha. and .beta. chains are both characterized by a pH-dependent spectral transition from a type I to a type II spectrum. The chains are non-equivalent with regard to both the type I and the type II spectra. The type I spectra assigned to the .alpha. and the .beta. chains are characterized by .**GRAPHIC**. = 2.0095 with a hyperfine splitting of .**GRAPHIC**. (15NO) = 2.36 mT and gzz = 2.0085 with a hyperfine splitting of azz (15NO) = 2.41 mT, respectively. The type II spectra assigned to the .alpha. and the .beta. chains are characterized by .**GRAPHIC**. = 2.005 and a hyperfine splitting of .**GRAPHIC**. (15NO) = 3.07 mT and .**GRAPHIC**. = 2.005 and a hyperfine splitting of .**GRAPHIC**. (15NO) = 3.31 mT. The change of the hyperfine splitting at gzz during the transition from type I to type II corresponds to an increase of the spin density at the NO by about 25% in both types of chains. Comparison of type I spectra of the NO-ligated .alpha. and .beta. chains, respectively, demonstrates that the spin density at the NO is larger in the .beta. chains than in the .alpha. chains. The spectral types are correlated with functional states defined by the kinetics of NO-binding. Binding of inositol hexaphosphate has no influence on the ESR spectra in the whole range of pH as it is expected if NO-Hb Kansas is in the quaternary T structure.