The purification, characterization and ligand-binding kinetics of hemoglobins from root nodules of the non-leguminous Casuarina glauca — Frankia symbiosis

Abstract

The presence of a membrane-bound hemoglobin in aqueous extracts of nitrogen-fixing Casuarina root nodules (Davenport, H.E. (1960) Nature 186, 653–654) has been confirmed. By strictly anaerobic grinding and extraction under carbon monoxide, with inclusion of soluble polyvinylpyrrolidone and zwitterionic detergent in extraction buffer, soluble carboxyhemoglobin was obtained. This was purified by anaerobic ‘adsorption’ chromatography on Sephacryl S-200 (Pharmacia) followed by aerobic molecular exclusion chromatography on Sephadex G-75 (Pharmacia) to yield very stable oxyhemoglobin. By preparative-scale isoelectric focusing Casuarina oxyhemoglobin is separable into three major components comprising approx. 20% of applied protein, and very many minor components. Monomeric Casuarina hemoglobin is similar to other plant hemoglobins in respect of molecular weight (≈ 17 500), optical spectra, extremely rapid kinetics of binding to oxygen and carbon monoxide and high oxygen affinity ( P 50 ≈ 0.074 torr). Hence, it is possible that this protein functions in the Casuarina symbiosis as does leghemoglobin in leguminous nitrogen-fixing symbioses. Western blot analysis showed close immunological relationships between the non-leguminous Casuarina and Parasponia hemoglobins and a weaker relationship between these two proteins and soybean leghemoglobin. It is proposed that these hemoglobins from widely separated plant orders have a common evolutionary origin.