Purification and Molecular Properties of Yeast Hemoglobin

Abstract

Yeast hemoglobin from Candida mycoderma has been purified by ammonium sulfate fractionation and subsequent DEAE‐cellulose column chromatography. A minimum molecular weight estimated from the result of amino acid analysis, as well as dodecylsulfate gel electrophoresis and gel filtration on Sephadex G‐100, is 50000. The absorption spectra of ferrous yeast hemoglobin in the oxy, deoxy and carboxy forms were almost identical to those of other hemoglobins, with the exception of a small contribution from flavin. The molecule has two prosthetic groups, FAD and protoheme. The results obtained by dodecylsulfate‐gel electrophoresis and 8 M urea treatment suggest that both prosthetic groups are attached on a single peptide chain, which appears to exist as a monomer. Yeast hemoglobin, in the ferrous form, is capable of binding oxygen reversibly. The oxygen affinity of yeast hemoglobin is 20 nM (0.01 mm Hg) and a rate constant of oxygen dissociation is 17 s −1 at pH 7.0, 23°C. Half‐reduction potentials of heme and flavin moieties were ‐60 mV and about ‐150 mV, respectively, in 0.1 M phosphate buffer pH 7.0 at 25°C.