Proton magnetic resonance studies of human cyanomethemoglobin.

Abstract

Human hemoglobin has a molecular weight of 64,500 and is composed of four subunits, two each of two types, a and F. Each subunit consists of an aor fpolypeptide chain and one protoheme IX group. In the biologically active state, each heme group contains one iron (II) ion which binds molecular oxygen in its first coordination sphere. The entire hemoglobin molecule, a232, thus contains four heme groups and binds four oxygen molecules in the fully oxygenated state. It has been fournd that the oxygen affinity is closely related to the structure of hemoglobins.' In the present paper we give a preliminary account of proton nuclear magnetic resonance (NMR) studies of human cyanomethemoglobin. From the NAMR data, electron spin densities at various positions in the heme group are derived. We indicate how this kind of N1\IR experiment can give new information about some aspects of the relations between structure and function in hemoglobin. In the NA/MR spectra of proteins, essentially all the proton resonances are observed in a narrow range which extends from the internal standard DSS (2,2dimethyl-2-silapentane-5-sulfonate) downfield to ca. -10 ppm (parts per million relative to DSS). The larger the protein molecule, the more the resoniances of the protons of the individual amino acid side chains overlap. Therefore most NMR studies are done with relatively small proteins with molecular weights less than 20,000. In paramagnetic heme proteins, local magnetic fields arising both from aromatic ring currents2' I and from the unpaired electron spins have been shown to shift certain proton resonances out of the range between DSS and -10 ppm.4-6 For these reasons the NMR spectrum of cyanomethemoglobin contains a considerable number of resolved resonances despite its high molecular weight. Experimental.-Human hemoglobin solutions were prepared from the freshly drawn blood of one of us (K. W.) by the following method. The erythrocytes were separated from the plasma by centrifugation within 15 min. after the addition of the anticoagulant (sodium oxalate), then washed four times with 1 vol of 1% NaCl. After hemolysis, the solution was subjected to 105,000 g centrifugation for 21/2 hr. The upper two thirds of the supernatant, free of any flocculus precipitate, was separated off and then further centrifuged. This procedure was repeated twice. The hemoglobin solution was dialyzed overnight at 4?C against 0.1 M phosphate buffer. Some hemoglobin solutions were also prepared by the widely used toluene method.7 The NMR spectra of cyanomethemoglobin solutions prepared by these two different methods were found not to differ noticeably. Methemoglobin was prepared by addition of a sixfold excess of K3Fe(CN)6 to the hemoglobin solution that was thenl dialyzed extensively against 0.1 M phosphate buffer. Methemoglobin was purified on the cation exchange resin Bio-Rex 70 (Bio-Rad) and concentrated by vacuum dialysis. Sodium phosphate buffer, pH 6.42, total ionic strength of 0.304, was used for the elution of methemoglobin.8 The homogeneity of isolated hemoglobin fractions was checked by starch gel electrophoresis9 with the discontinuous buffer system.10 Methemoglobin A1 was then converted to cyanomethemoglobin by the addition of a freshly prepared solution of KCN in phosphate buffer.