Studies on the Mechanism of Action of Cyanate in Sickle Cell Disease

Abstract

Abstract Tetramers of hemoglobin S carbamylated on the NH2-terminal residues of the α chains (α2cβ2), the β chains (α2β2c), or both chains (α2cβ2c) have been prepared. For this purpose carbamylation was carried out with deoxyhemoglobin to yield a product that was completely modified at the NH2-terminal residues and minimally carbamylated at lysine residues. The carbamylated α or β chains, separated and purified as the p-hydroxymercuribenzoate derivatives by ion exchange chromatography, were recombined with equimolar amounts of the carbamylated or the uncarbamylated p-hydroxymercuribenzoate chains in the presence of a 300- fold-excess of 2-mercaptoethanol at 4° to give a quantitative yield of hybrid hemoglobin tetramer; regeneration of the —SH groups prior to mixing was not necessary. Each hybrid gave a single band on gel electrophoresis and showed normal SH reactivity upon titration with p-hydroxymercuribenzoate. The log p50 values of the samples (0.2 mm in heme, and free of organic phosphates), in 0.1 m Tris-Cl (total Cl-, -0.1 m), pH 7.4, and 25°, were 0.35, 0.41, 0.59, 0.61, and 0.68 for α2cβ2, α2cβ2c, hybrid α2β2, native α2β2 and α2β2c, respectively. Hill coefficients for the samples were between 2.6 and 3.1. The minimum gelling concentrations at 25° for deoxygenated α2cβ2, α2cβ2, hybrid α2β2, native α2β2c were 23.5, 29.1, 23.9, 24.1, and 29.4 g per 100 ml, respectively. These results show that carbamylation of the α chains increases the oxygen affinity but does not affect the minimum gelling concentration of hemoglobin S. Complete carbamylation of the β chains decreases the oxygen affinity and leads to an increased minimum gelling concentration. As previously reported for patients on oral cyanate therapy at low levels of carbamylation, the α chain is carbamylated almost twice as much as the β chain in vivo and the oxygen affinity of their red cells is increased. These results, taken together with the present finding that the increase in the minimum gelling concentration of α2cβ2c is modest, suggest that the inhibition of cell sickling by cyanate in vivo is mediated primarily through the increased oxygen affinity of carbamylated hemoglobin S.