Exclusion chromatography of concentrated hemoglobin solutions. Comparison of the self-association behavior of the oxy and deoxy forms of the alpha 2 beta 2 species.
Theory pertaining to the interpretation of partition chromatography results obtained with self-associating protein systems studied at high total concentrations is extended to permit consideration of situations in which both monomeric and dimeric states partition. This development, which includes considerations of thermodynamic nonideality effects, permits a quantitative correlation of human oxyhemoglobin results reported previously and obtained in this work employing a different stationary matrix of controlled-pore glass beads. The two sets of results, obtained at pH 7.3 and 20 degree C, indicate that the alpha 2 beta 2 species of oxyhemoglobin self-associates. Two types of association pattern, discrete dimerization and an indefinite self-association, are examined. This is done for a realistic range of values for the radius, r. of the effective hard sphere appropriate to the calculation of the covolume of the alpha 2 beta 2 species in the assessment of the thermodynamic nonideality contribution. Assessed values of the isodesmic association constant range from 66 +/- 23 M-1 (r - 2.84 nm) to 154 +/- 26 M-1 (r = 3.13 nm). This mode of indefinite association is marginally favored over dimerization when the larger value of r is considered, the two patterns becoming virtually indistinguishable for the lower value of r. Partition chromatography results are also presented for human deoxyhemoglobin up to a total concentration of 225 g/l, and are analyzed in a similar fashion to show that the indefinite self-association pattern is favored, governed by an isodesmic constant in the range 91 +/- 9 M-1 (r = 2.84 nm) to 223 +/- 84 M-1 (r = 3.13 nm). Comparison of the constants assessed for the oxy and deoxy systems permits discussion of the concept that oxygen binds preferentially to the alpha 2 beta 2 species of deoxyhemoglobin in comparison with its polymers.
D002850:Chromatography, Gel / Q000379:methods; D006454:Hemoglobins; D006801:Humans; D046911:Macromolecular Substances; D008433:Mathematics; D010108:Oxyhemoglobins
D006454:Hemoglobins; D046911:Macromolecular Substances; D010108:Oxyhemoglobins