An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t1/2 using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L [14C]leucine indicated a t1/2 of 7.2 days. When stroma free hemolysates were heated at 50° C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37° C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant β chain by precipitation with p hydroxymercuribenzoate. Purification of the β chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for β32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at β31 to impair heme stabilization.
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