A laser light-scattering study of haemoglobin systems

Abstract

To investigate haemoglobin systems by light scattering, it is necessary to use wavelengths at the red end of the spectrum where absorption is low. Accordingly, a helium-neon laser has been used and a light scattering apparatus has been designed and constructed around it. The light scattering envelope for bovine serum albumin solutions has been used as a test of the apparatus and its alignment. Calibration has been performed against accurate spectrophotometric measurements using stable dispersions of colloidal silica. Both human and sheep haemoglobin samples have been investigated at neutral pH and at a series of concentrations up to 0.6 g/100 ml. The CO-derivatives of the two haemoglobins behave similarly. At low ionic strength ( I = 0.1), dissociation is small with a dissociation constant not greater than 1 x 10 –6 mol/l. At higher ionic strength (2 M NaCl), dissociation is much more marked with a dissociation constant of ca . 3 x 10 –5 mol/l. These values are in fair agreement with previously published estimates from this laboratory based upon both osmotic pressure and sedimentation equilibrium studies. They are also in substantial agree­ment with most of the estimates made by other workers using a range of techniques, but in considerable excess of those reported by one group of workers (Cassassa and co-workers) using laser light scattering. In a later paper, however, this same group revised their values upwards. Sheep deoxy haemoglobin was found to dissociate to a smaller, almost undetectable, extent at low ionic strength but to an appreciable extent ( K = 1 x 10 –5 mol/l) in 2 M NaCl. The similarity in dissociation behaviour of sheep and human haemoglobins suggests similarity in the amino acid composition at those interfaces of the αβ subunits which are responsible for association.