Hemoglobin Philly (beta 35 tyrosine phenylalanine): studies in the molecular pathology of hemoglobin.

Abstract

An abnormal unstable hemoglobin, hemoglobin Philly, was found in three members of a family, each of whom had evidence of a chronic hemolytic state. The presence of the mutant protein was suggested by the rapid appearance of inclusion bodies upon incubation of erythrocytes with brilliant cresyl blue and by the increased heat precipitability of the hemoglobin. However, no abnormal hemoglobin could be demonstrated by electrophoresis or column chromatography. Sulfhydryl titration of the hemolysates with p-mercuribenzoate indicated that there was an average of four reactive sulfhydryl groups per hemoglobin molecule instead of the usual two. The total number of hemoglobin sulfhydryl groups was normal; six groups were measured when denatured globin was reacted with 5,5'-dithiobis[2-nitrobenzoic acid]. This indicated that the increased sulfhydryl reactivity was due to an increased availability to p-mercuribenzoate of the usually unreactive hemoglobin cysteines at beta112 and alpha104. After treatment for (1/2) hr with 4-5 moles of p-mercuribenzoate per mole of hemoglobin, electrophoresis revealed that 30-35% of the hemoglobin had been dissociated into alpha- and beta-chains. Normal hemolysates revealed negligible splitting after 72 hr of similar treatment. The alpha- and beta-chains of hemoglobin Philly were separated from the unsplit hemoglobin A by carboxymethyl cellulose chromatography. Fingerprint and amino acid analyses revealed that tyrosine beta35 was replaced by phenylalanine. In hemoglobin Philly there is loss of the normal hydrogen bond between the tyrosine hydroxyl group and the carboxyl group of aspartic acid alpha126 at the alpha(1)beta(1) contact. This shifts the equilibrium from hemoglobin tetramers toward monomers, exposing the beta112 and alpha104 cysteines. In the cell, precipitation of the unstable monomers may contribute to erythrocyte destruction.