Nitrogen binding to deoxyhemoglobin at high pressures and its relation to changes in hemoglobin-oxygen affinity.

W A Gerth

doi:10.1016/s0021-9258(18)68272-9

Abstract

The stoichiometric and thermodynamic properties of nitrogen (N2) binding to human deoxyhemoglobin (Hb) at N2 saturation pressures up to 400 atm were derived from measured N2 solubilities in protein-free buffers (pH 7.1) and in corresponding buffer + Hb (6.5% w/w) solutions at 20.0, 25.0, and 37.0 degrees C. At each temperature, approximately 3 N2 molecules bind per Hb tetramer at N2 pressures of 100 atm, while about 7 N2 molecules bind per tetramer at 400 atm N2 pressure, where available binding sites are still not fully saturated. Calculated N2-Hb binding isotherms are well described by a simple binding model with 12 independent and equivalent binding sites/Hb tetramer. N2 binding at each of the sites is hydrophobic, exhibiting the typical increase of the dissociation enthalpy with temperature. Enthalpies of dissociation are slightly more negative, while corresponding unitary entropies are somewhat less negative than those for the transfer of N2 from olive oil to water. Calculated partial molar volumes of N2 in Hb are positive but less than the corresponding partial molar volumes of N2 in buffer. Results indicate that N2 binding to Hb is accompanied by only small protein conformational changes which entail slight structural destabilization and loss of free volume in the protein that partially accommodates the volume of the N2 ligand. Results are related to previously reported effects of high pressure and high-pressure N2 on HbO2 affinity, illuminating essential features of the molecular mechanisms for these effects.

Highlights

At Nz saturation pressuresup to 400 atm were derived from measured Nz solubilities in protein-free buffers and in corresponding buffer+ Hb (6.5%w/w) solutions at20.0, 25.0,and 37.0 “C. At each temperature, approximately 3 Nz molecules bind perH b tetramer at Nz pressures of 100 atm, while about 7 NZ molecules bind per tetramer at 400 atm NZpressure, where available binding sites are still not fully saturated.Calculated Nz-Hb bindingisotherms are well described by a simple binding mwoidtehl 12 independent and equivalent binding sites/Hb tetramNeZr.binding at each of the sites is hydrophobic, exhibiting the typical increaseof the dissociation enthalpywith temperature
In washed intact erythrocytes at a given pressure, the N2 effect on hemoglobin-oxygen ( H b 0 2 ) affinity is larger than that of the less soluble helium, and the effects of either high pressure gas are fully reversible with decompression to 1atm [8].The nature of the high pressure gas effect is to rendernormal sigmoidally shapedHbOn saturation curves hyperbolic and similar in form to those for myoglobin, suggesting that the gas-induced increases ofHbO2 affinity occur by a mechanism that impairs allosteric interactions between different subunits of the Hb tetramedruring oxygenation
Various nonmetabolic atmospheric and anestheticgases are “enough to change our effect from an increase to a decrease known to hydrophobically bind hemoglobin [1,2,3,4,5,6] and cause in affinity. .. .”More recent work has firmly established that reversible and often specific alterations of its molecular struc- the partial molar volume of O2 is positive in aqueous liquids ture [6, 7]

Summary

Introduction

Nitrogen-Hb binding isotherms were calculated using Equation 4 with the fitted equations [2] for the Sg functions in order to exploit the applicability of Henry's law to the NZ solubilities in the protein-free buffers.

Results

Conclusion