The Structure of Hemoglobin Indianapolis [P112(G14) Arginine]

Hemoglobin Indianapolis is an abnormal human hemoglobin whose structure could not be deduced by standard techniques because of its extreme lability. This variant hemoglobin was found in two patients with the phenotype of severe jl-thalassemia. An unstable variant hemoglobin could not be demonstrated by any of the standard techniques of hemoglobin identification. Biosynthetic studies revealed an imbalanced /3/a-globin chain ration of 0.4, typical of heterozygous /l-thalassemia. However, an anomalous peak of radioactivity, with no corresponding absorbance at 280 nm, was present in the chain separation chromatograms. The proportion of the aberrant peak of radioactivity progressively decreased as incubation time increased, and it was totally abolished if the hemolysate was heated to 50°C prior to chain separation. These findings suggested the presence of an extremely labile fi chain which was degraded shortly after synthesis. Normal /3 chains were labeled with L-[U-‘%Jleucine and mixed with the abnormal peak labeled with L-[4,5-3H]leucine. The tryptic peptides of this mixture were separated by column chromatography and indicated the presence of an abnormal j3 chain with an amino acid substitution in /?T-12a. A qualitative amino acid composition of this peptide was obtained by labeling the peptide with a mixture of 3H-amino-acids. These data, along with other evidence, indicated that the structure of the variant /I chain was /l112(G14) Cys --) Arg. The new arginine at this position would be expected to cause repulsion of the B and G helices, causing the instability of the molecule.