pH DEPENDANT XANES OF CARBONYLHEMOGLOBIN BY FAST DISPERSIVE SPECTROSCOPY

Fast x-Ray dispersive spectroscopy allowed by the dispersive geometric shows three transitions in the kinetics of 5mM carbonylhemoglobin denaturation. The CO detachment at low pH(2.6) is preceeded by CO bending due to the protonation (pH-5.6), and later by protein unfolding (pH-3.3) Only a few detection systems have been achieved to compete the creditable performance of synchrotron radiation source. In the X-Ray absorption field, the combination of a well-known X-Ray energy dispersive optics and a position, sensitive detecror able to work under high flux conditions enlarges the scope of this spectroscopy. This paper, devoted to a pH dependant XANES study of the pH-denaturation of carbonylhemoglobin offers the opportunity to discuss the advantages brought by the dispersive scheme applied to biophysics. The ion XANES spectra shown in fig. 1 has been recorded using a Si 31 1 to give an account of the energy resolution which is as good as it is with a step by step scan using a double detuned crystal. In addition, because of the lack of mechanical movements, once the optics is tuned for a given absorption edge, the energy scale is found absolutely stable. The in-situ observation permits a systematic investigation of the time-dependant system and an a-posteriori decision about stages of interest. Anticipating the kinetics discussed below we would like to indicate that we bin the spectra by set of 10 or 5 according to the importance of the change observed in the relevant PH-domain PH dependant XANES of 5 mM carbonylhemoglobin solution. Knowledge of Fe-C-0 and Fe-02 bond angles in solution is fundamental to the understanding of the reversible molecular binding of diatomic molecules in haem proteins. XAPdES has been used sucessfully to such determination for protein crystals as well as solutions.(2) Different approaches including infrared CO stretching, 13c nuclear magnetic resonance, optical absorption try to correlate the CO bending or tilting with respect ot the porphyrin plane to a steric constraint either from the distal side of the heme pocket or from the proximal side. Recent improvements in XANES calculations performed at Rome, including superior multiple scattering treatment confirms the validity of the assignment of the 16 eV resonance to CO bending tilting It is this resonance which will be used as a probe for the present study which is concerned with the pH denaturation of the heam protein. The time dependance is used to ramp down the pH value. Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868236 C8-1202 JOURNAL DE PHYSIQUE