On the dissociation of the sheep haemoglobin molecule at neutral pH I. Osmotic pressure measurements

Abstract

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.