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The Yphantis method of high-speed sedimentation equilibrium has been used to investigate the dissociation of sheep haemoglobin (type B) at low protein concentration (0.01 to 0.1 g/dl) in both NaCl (buffered and unbuffered) and phosphate buffer solutions at approximately neutral pH, over a range of ionic strength, temperatures of 5 and 25°C being employed. The importance of reliable ‘blank’ solvent control experiments was clearly demonstrated experimentally and weight average molecular weight data were shown to be more reliable than number averages. Apart from work at 25°C and high ionic strengths, the results are compatible with a reversible tetramer-dimer ( α 2 β 2 ⇌ 2 αβ ) dissociation. The dissociation constants obtained showed considerable agreement with those determined by osmotic pressure (reported in the previous paper) for the lower ionic strengths ( I < 1.0), but at 2 mol/l NaCl and 5°C, the sedimentation equilibrium value was 40 % higher. This probably arises through the preferential uptake of water from the high ionic strength solvent, though some dimer-monomer ( αβ ⇌ α + β ) dissociation cannot be completely excluded. On the other hand, in 2 mol/l NaCl at 25°C, the presence of monomeric units was strongly indicated, in conformity with osmotic pressure and other indications. At low NaCl concentrations, the dissociation was not significantly affected by a temperature increase from 5 to 25°C, but in phosphate solutions of comparable ionic strength, significantly less dissociation occurred and an enthalpy increase on dissociation of 55 to 63 kJ/mol was observed.

The stability of the sheep haemoglobin molecule (type B) has been investigated at neutral pH over a range of ionic strengths by osmotic pressure ( π ) measurements in phosphate buffer and unbuffered NaCl solutions at 5 and 22 °C. At low ionic strength ( I < 0.10), no significant dissociation of the molecule could be detected and the molecular weight obtained ( M = 64 100) was in good agreement with that obtained from amino acid sequence studies (64 500). At higher ionic strength ( I > 0.10), reversible dissociation was detectable as a deviation from a straight line plot of π/c against c , and, using a graphical method as well as a computer program, the most probable values of the dissociation constant ( K d ) (describing the dissociation, α 2 β 2 ⇌ 2 αβ ) and interaction constant ( B' ) were obtained. The dissociation constants increased significantly with increasing ionic strength up to I = 2, but thereafter remained approximately constant. A plot of the standard free energy of dissociation,∆ G 0 d , against ionic strength was curved but extrapolated to a value of approximately 32 kJ /mol in good agreement with that found for human haemoglobin in the presence of a variety of dissociating reagents. At the higher ionic strengths ( I = 2) and particularly at 22 °C, there was some evidence that dissociation proceeded further than αβ but further work is required to substantiate this finding.

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