Reactions of partially oxidised hemoglobins solution. III

Abstract

A sheep oxyhemoglobin of the B type was partially oxidised by ferricyanide and subjected to ion-exchange chromatography on CM-Sephadex. A fraction, F, was isolated containing hemoglobin derivatives in which two iron atoms were in the ferric state. A comparison of the reactions of F with those of solutions prepared either by randomly oxidising oxyhemoglobin, R, or by prior admixture of methemoglobin with oxyhemoglobin, PA, showed that: 1. 1. In dilute buffers at pH 6.1, the reactions of F or oxyhemoglobin with CO were not distinguishable. 2. 2. In dilute buffer at pH 7.0, the initial rate constants for the reaction of CO with deoxygeneted solutions of R increased with the degree of oxidation when measured at 30°: these changes were associated with an increase in activation energy. The corresponding reaction of F showed kinetic properties characteristic of R when compared at the same overall composition. When solutions of hemoglobin and methemoglobin were mixed at the same time that reaction with CO was initiated the resulting reaction was typical of hemoglobin but when deoxygenated solutions of PA were prepared in advance their properties were identical with solutions of R of the same composition. 3. 3. In dilute buffer at pH 5.1 or in solutions of 2 M NaCl at pH 7, the reaction of CO with deoxygenated solutions of R was less dependent on the degree of oxidation. In these conditions, the properties of F were characteristic of highly oxidised samples of R, whereas, when PA was prepared in solutions of high ionic strength, its properties resembled those of R with lower degrees of oxidation.