Biophysical Journal 55: 703-712 (1989)
© 1989 the Biophysical Society

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Detection of the heme perturbations caused by the quaternary R----T transition in oxyhemoglobin trout IV by resonance Raman scattering.

University of Bremen, Physics Department, Federal Republic of Germany.

ABSTRACT

The depolarization ratio dispersion and the respective excitation profiles of two structural sensitive Raman lines of oxyhemoglobin-trout IV (1,375 and 1,638 cm-1) have been measured at pH-values between 6.5 and 8.5. They were analyzed by employing a fifth order time dependent perturbation theory to calculate the polarizability tensor. This provides information about the pH-dependence of parameters reflecting symmetry classified distortions of the prosthetic heme groups. In order to correlate these distortions with functional properties of the molecule the following protocol has been employed: (a) a titration model was formulated relating each conformation of the molecule to a distinct set of distortion parameters the incoherent superposition of which provides the respective distortion parameter obtained from our Raman data. (b) The thermodynamic constants determining the equilibrium between these molecular conformations (i.e., the quaternary T and R-states, the low affinity t and the high affinity r-states of the distinct subunits, the pK-values of the Root- and Bohr groups) were obtained from a set of O2-binding curves that were analyzed in terms of an allosteric model suggested by Herzfeld and Stanley 1974. J. Mol. Biol. 82:231. The application of this procedure yields excellent reproduction of the pH-dependent effective distortion parameters of both Raman lines investigated. Thus established correlation between hemoglobin function (O2-binding) and structure (asymmetric perturbation of the hemegroup) provides some interesting insights into the molecular basis of the allosteric Root effect.