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Hemoglobin variants with two amino acid substitutions affecting one globin chain are relatively rare. Hb T-Cambodia, a doubly substituted beta-globin variant, was characterized previously by amino acid sequencing as having sequence alterations in beta 26 (beta 8)Glu-->Lys and beta 121(GH4) Glu-->Gln (1). It is a variant that migrates cathodic to Hb A2 on alkaline electrophoresis and with Hb A on acid citrate agar electrophoresis. We report here the mutations of Hb T-Cambodia at the nucleotide level using DNA sequencing, in beta-globin gene codon 121 (GAA-->CAA) and in codon 26 (GAG-->AAG). These are the mutations of Hb D-Punjab and Hb E, respectively.

Using a TRS-80 Radio Shack microcomputer system, we have computerized the differentiation of hemoglobin variants according to their mobilities in four methods of zone electrophoresis, each measuring a somewhat different aspect of molecular structure. In the identification routine of the program, the electrophoretic mobilities of an unknown variant are compared mathematically to the mobilities of previously analyzed variants; the computer prints out those known variants whose mobilities are within a specifiable interval on either side of the mobilities of the unknown variant. Properly specifying this interval is essential to the effectiveness of the identification. Another routine in the program classifies the variants according to any of ten molecular variables. Computerizing the identification and the information output provides a way to differentiate many variants, allows convenient handling of a large number of data on them, and facilitates their structural analysis.

Hb Long Island was found in a diabetic man and his nondiabetic mother as the result of a routine clinical measurement of Hb AIc. It is present in amounts approximately equal to Hb A. Its alpha chains are normal but its beta chains have two alterations compared to the normal. A methionyl residue is attached to the usual NH2-terminal valyl residue. This valyl residue is followed by prolyl residue in place of the usual histidyl residue 2. The remaining sequence of the beta chain is normal. No hemoglobin or abnormal beta chain containing only the prolyl substitution could be detected by several different electrophoretic and HPLC procedures. We postulate that Hb Long Island is the result of a mutation in which a single nucleotide change causes the substitution of a prolyl residue for the normal histidyl residue at position 2 of the beta chain. We further postulate that this abnormal prolyl residue inhibits enzymatic cleavage of the initiator methionyl residue from the abnormal beta chain during posttranslational processing. Although the oxygen affinities of the whole blood, suspended cells, and hemolysate are normal, the affinity of the isolated Hb Long Island is slightly decreased and the effects of organic phosphates are reduced compared to normal. These changes are consistent with the loss of the normal histidyl residue 2 and the extension of the NH2-terminal end of the beta-chain molecule.

A system of calculating relative mobilities of mutant hemoglobins and globin chains in four methods of zone electrophoresis is described. In electrophoresis on cellulose acetate, TEB buffer, pH 8.5, mobilities are calculated as ratios of the mobility of simultaneously analyzed Hb C. In electrophoresis on citrate agar, pH 6.0, anodic mobilities are also related to Hb C, cathodic ones to Hb F. In globin electrophoresis in urea 2-mercaptoethanol buffers, pH 6.0 and 8.9, mobilities of the mutant globin chains are calculated in relation to the mobilities of normal alpha and beta chains. These calculations provide objective comparisons of mobilities of hemoglobins, including those analyzed at different times. The combined data permit recognition of many mutant hemoglobins.