Separation of Carboxy- and Methemoglobin by Cation Exchange Dextran Gels

Abstract

Human adult carboxyhemoglobin (Hb,-Co), methemoglobin (MHb,), and mixtures of the pure derivatives were transferred into phosphate buffer pH 6.0 by gel filtration on Sephadex G 25 and then adsorbed on two different ion exchangers with a high degree of swelling. In one case the charged groups were carboxyl, CM-Sephadex C-50, and in the other sulphonate, SE-Sephadex C-50. They were then eluted from the column with a continuously increasing concentration of NaCl in the same buffer.

On CM-Sephadex, Hb_A CO separated into two fractions, probably HbA₀, CO and HbA₁CO, but this fractionation was poor on SE-Sephadex.

Mixtures of Hb, CO and hfHb, gave a good separation on SE-Sephadex, less good on CM-Sephadex. A two-step elution procedure gave a good separation on SE-Sephadex with purities of over 90 per cent in parts of both fractions. The elution behavior indicated that the same result can be obtained by elution with a constant eluant concentration.