Mechanism of inhibition of hemoglobin S polymerization by cyanate

We have studied the effect of carbamylation on hemoglobin S (HbS) polymerization with the use of a new quantitative gelling technique. HbS gels fromed in the presence of nitrogen and dithionite (with or without carbon monoxide) at pH 7 in 0.15M phosphate were separated into sol and gel phases by centrifugation at 130,000 g. Hb concentration to the sol phase ([Hb]sol) of nonligated HbS was found to be constant (10.5 +/- 092 mM heme) over a range of original Hb concentrations from 11 to 17 mM at 24degrees C. This suggests that the HbS sol-gel equilibrium behaves as simple two-phase system. Increasing levels of total carbamylation from 0.65 to 3.65 moles CNO-/mole Hb tetramer (0.36 to 2.2 moles N-terminal/mole Hb4) progressively increases [Hb]sol. Specific activity of 14CNO-HbS was similar in the sol and gel phases, whereas COHbS appeared to be completely excluded from polymer structure of the gel. A comparison of the solubility of uncarbamylated and heavily carbamylated HbS at Co saturations ranging from 3 to 61 percent showed that the larges difference in [Hb]sol occured at the lowest ligand saturation rather than at intermediate states of ligation. Inhibition of HbS polymerization by carbamylation under these conditions, therefore, is not primarily the result of an effect on the T in equilibrium R comformational equilibrium. Our findings indicate that cyanate can interfere with HbS polymerization directly, possibly by alteration of surface binding sit(s) which are critical to aggregation. This direct action of cyanate may contribute significantly to the hematologic improvement achieved by cyanate treatment in sickle cell disease.