Methylisocyanate as an antisickling agent and its reaction with hemoglobin S

Reviewing the reaction of potassium cyanate, an antisickling agent, with alpha-amino groups of hemoglobin, it was found that the reaction was a slow process and requires a large excess of the reagent. The reason for the slow reaction rate of carbamylation of hemoglobin by cyanate is that cyanate itself does not react with hemoglobin. It is rather isocyanic acid, the reactive species, that reacts with hemoglobin. Since the pK of isocyanic acid is 3.8, only one out of 4,000 cyanate ions is present as isocyanic acid at physiological pH. Therefore, it appears that a large excess of cyanate ions is required to achieve the carbamylation of hemoglobin S, both in vivo and in vitro. Furthermore, the pH optimum for carbamylation of carboxyhemoglobin and deoxyhemoglobin is 5.5 and, at pH 7.4, the reaction velocity drops to one-third for carboxyhemoglobin and one-half for deoxyhemoglobin. To seek an approach to reduce the dosage of cyanate and to increase the reaction velocity, an isocyanate derivative, methylisocyanate which is already in the reactive form, was tested for its antisickling activity and its reaction with hemoglobin S. It was found that methylisocyanate had antisickling activity and that only a stoichiometric amount to 2-fold excess of the reagent over hemoglobin S alpha-amino groups was required to prevent the sickling of erythrocytes. Methylisocyanate-treated sickle erythrocytes showed an increased oxygen affinity compared to untreated methylisocyanate reacted with alpha-amino groups of hemoglobin S and the reaction was complete in less than 1 min. Methylcarbamylated hemoglobin S had a higher minimum gelling concentration than the untreated hemoglobin S. There was no detectable reaction of free sulfhydryl and epsilon-amino groups of hemoglobin S with methylisocyanate. These results indicate that methylisocyanate, and probably other isocyanate derivatives, possesses powerful antisickling activity.