Resonance Raman spectroscopic evidence that carp deoxyhemoglobin remains in a T-like quaternary structure at high pH: implications for cooperativity

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Abstract
Resonance Raman spectroscopy shows the Fe-proximal imidazole stretching band to shift from 215 to 219 cm-1 between human deoxyhemoglobin (deoxy-Hb) and a Hb sample which is 75% oxygenated, demonstrating that the T-R quaternary structure switch can be monitored by resonance Raman spectroscopy in native Hb at equilibrium. For deoxy-Hb from carp, the band is at 215 cm-1 at pH 9 as well as pH 6, contrary to previous reports of an elevated frequency at high pH. The invariance of this frequency over a large affinity difference is in contrast to a recent report of continuously varying vFe-ImH frequencies for human mutant deoxy-Hb''s. The band shifts to 219 cm-1 for carp Hb and pH 9 when O2 is bound to only 20% of the hemes. The spectra are consistent with a T-R switch upon binding .apprx. 0.5 O2 per Hb on the average, although the number may be higher if the binding affinity is higher for .alpha.- than for .beta.-chains. The 0.5 value, in conjuncation with the weak cooperativity observed for carp Hb at pH 9, is incompatible with a value of the allosteric constant, L = (T0)/(R0), large enough to prevent the vFe-ImH band from shifting detectably at pH 9 in the absence of O2. The possibility of functionally important intermediate structures is discussed.

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