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

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 (Kd) (describing the dissociation, α2β2 ⇌ 2αβ) 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,∆G0d , 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.