Side reactions in the deoxygenation of dilute oxyhaemoglobin solutions by sodium dithionite.

Sodium dithionite (Na2S204) is the commonest and most convenient reagent for the deoxygenation and reduction of haem pigments. The danger of undesirable side reactions between haem pigments and Na2S204 or its oxidation products has been remarked by several workers (Hartridge & Roughton, 1923; Keilin, 1926, 1927; Zeile, 1932; Lemberg, Legge & Lockwood, 1941; Lemberg & Legge, 1949), and is particularly important in connexion with accurate kinetic studies of the deoxygenation of oxyhaemoglobin (HbO2) in an excess of Na2S204. Hartridge & Roughton (1923) studied the deoxygenation of HbO2 by Na2S204 in their rapid-flow apparatus, and concluded that the Na2S204 did not react directly with the HbO2, but simply removed the oxygen from the solution as fast as it was liberated, and that the direct and quantitative conversion of HbO2 was by a simple first-order dissociation. These conclusions seemed to be confirmed by the more accurate and comprehensive measurements of Millikan (1933, 1936). Recently, however, evidence of a more complicated state of affairs was obtained by Legge & Roughton (1950), both in a re-inspection of Millikan's results and in new experiments on sheep HbO2 by a similar method. First, it appeared that the first-order velocity coefficient increased significantly during the first third of the reaction, as was indeed to be expected on the basis ofthe intermediate-compound theory of haemoglobin (Hb) reactions (Roughton, 1949). Secondly, in some experiments at neutral pH the last stages ofthe deoxygenation seemed to be extremely slow, and to be associated, at least in part, with the formation of pigments in side reactions brought about by Na2S204 oxidation products, which would invalidate the colorimetric method of analysis. The matter was not fully worked out, but it was suggested that a precursor of choleglobin might be the interfering substance. An initial increase of the first-order velocity coefficient has also been recorded for the deoxygenation of human HbO2, but no slow terminal stage was observed (Dalziel, 1954). Duringincidentalmeasurements, however, it was also noted that a perfectly stable Hb spectrum was not at once obtained on the addition of excess of Na2S204 to a dilute, slightly alkaline HbO2 solution in the absence of air, and that similar slow spectral changes in the absorption spectrum of a dilute Hb-Na2S204 solution were induced by briefexposure to air. These observations were consistent with the conclusions of Legge & Roughton (1950) that oxidation products of Na2S204 could bring about side reactions with the pigment. The purpose of the experiments described here was to obtain evidence of the nature of these side reactions by a closer examination of the slow spectral changes which followed the deoxygenation of HbO2 by Na2S204, and the action of oxygen on a Hb-Na2S204 solution, under various conditions of concentration and pH.