Préparation d'un tétramère dihéminique dérivé de l'hémoglobine

Abstract

The preparation and partial characterization of an unusual hemoglobin derivative, containing only 2 hemes per 4 chains, are described. The compound has been isolated in small yield from a solution of methemoglobin having undergone a partial loss of heme through transfer to apomyoglobin. The reaction mixture consists intially of methemoglobin and apomyoglobin, 6 mg/ml of each, at pH 7. After about half the total heme has been transferred to apomyoglobin, the reaction products are separated by fractional precipitation with ammonium sulfate. At 62% saturation, large amounts of apohemoglobin precipitate, together with small but significant amounts of a hemoprotein having solubility characteristics different from those of methemoglobin. The hemoprotein has been purified successively by gel filtration, CM-Sephadex chromatography and electrophoresis until it satisfied reasonably good criteria of purity. Sedimentation coefficients and molecular weight determinations accord well in demonstrating that the hemoprotein is a tetramer. Simultaneous analysis of iron and amino acids reveals a two-fold excess of amino acids over iron as compared with normal hemoglobin. The visible spectrum resembles that of methemoglobin in the relevant ionization state; the absorbance in the ultraviolet region (280 mμ) is, however, about twice as great as for methemoglobin for equal Soret band absorbance. The hemoprotein binds an amount of heme equivalent to that already present. We have called this di-heme derivative of hemoglobin semi-hemoglobin.