Testing the two-state model: anomalous effector binding to human hemoglobin

Abstract

Three allosteric states are required to describe the relaxation of (carbon monoxy) hemoglobin following flash photolysis. Combined absorbance and fluorescence probes were used. The absorbance signals consist of a component corresponding to ligand recombination and a component for the R-T transition. The fluorescence of 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), an analogue of 2,3-diphosphoglycerate, shows rates and amplitudes correlated with the absorbance transients. Measurements were made at pII 6, 6.5, and 7.0 at CO partial pressures of 0.1 and 1 atm. The fractional photolysis was varied in each case to change the initial distribution of the R states. Data show an immediate absorbance change due to ligand dissociation, while the changes in the ligand isosbestic and the fluorescence signals occur with time constants of 80 microseconds (at pH 6.5). The signals then show a biphasic return to equilibrium, characteristic of the allosteric system. The measurements provide three independent probes of the kinetics of the substates of hemoglobin. Although the ligand binding data can be generally represented by a two-state model, the fluorescence data require T states with different affinities for HPT.

Molecular Level Probing of Preferential Hydration and Its Modulation by Osmolytes through the Use of Pyranine Complexed to Hemoglobin

“…The extension to proteins via binding to HbA is a significant advance that permits the probing of the hydration shell at a functionally important site of a well studied protein. This advance is possible by virtue of the DPG-like binding properties of HPT (21)(22)(23)(24)(25) and the current finding that the wavelength of the fluorescence emission spectrum of HPT is sensitive to whether the HPT is bound to HbA or free in solution. This latter feature is important in providing assurance that for the HbA containing samples, the reported changes in the polyol/water ratio originate from HbA-bound HPT and not HPT that was released subsequent to the addition of reagents.…”

“…In the present study this sensitivity is exploited to probe: (i) the extent to which added glycerol invades the hydration shell of both free and HbA-bound HPT, and (ii) modulation of this effect as a function of added osmolytes including PEG, urea, trehalose and Mg 2ϩ . The potential utility of the HPT fluorescence as a probe of hydration is further enhanced by the observation that HPT, a known fluorescent analog of DPG, binds stoichiometrically to the DPG binding site of human adult hemoglobin (HbA), which is located at the ␤␤ terminus of the water-filled central cavity running through the central core of the ␣ 1 ␤ 1 /␣ 2 ␤ 2 HbA tetramer (21)(22)(23)(24)(25). This site is on the periphery of the protein, where the bound HPT is in contact with the hydration shell waters of HbA.…”

Probing the Hemoglobin Central Cavity by Direct Quantification of Effector Binding Using Fluorescence Lifetime Methods

“…Utilizing the observation that the fluorescence signal from HPT is highly quenched when bound to HbA, they performed steady-state fluorescence intensity measurements and established that HPT has a lower affinity for the DPG-binding site than DPG or IHP. A subsequent study by that group (11), using HPT as a probe of the DPG-binding site, focused on the detection of ligand-binding intermediates occurring along the R to T transition pathway. In those initial experiments, the results were limited in large part by two aspects of the methodology.…”

A potential determinant of enhanced crystallization of HbC: spectroscopic and functional evidence of an alteration in the central cavity of oxyHbC

“…First, the potential alterations of the central cavity of oxyHbC [known to be normal in deoxyHbC at ϳ5 Å resolution (Fitzgerald & Love, 1979)] were probed by investigating the relative binding of IHP and its functional effects in a comparative study with HbA. These studies utilized the fluorescent 2,3-DPG analogue, 8-hydroxy-1,3,6-pyrene trisulphonate (HPT) first used by Gibson and colleagues (MacQuarrie & Gibson, 1971Marden et al, 1986), and conventional oxygen equilibrium studies. Secondly, we explored the influence of central cavity ligands on the capacity of oxyHbC to crystallize, based on the notion that the ligands will stabilize the altered conformation, in a way reminiscent of the effect of IHP in the stabilization of the T state (Ogawa & Shulman, 1972;Cassoly & Gibson, 1972;Ackers et al, 1992;Mukerji & Spiro, 1994).…”