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Analysis of globin chains by reversed phase high performance liquid chromatography, used as an additional tool for characterizing hemoglobin variants, has led to the discovery of a new class of variants that display only differences in hydrophobicity. Two such variants are here described. Hb Ernz was found in a man of Italian origin who was polycythemic, and in two of his three daughters who were hematologically normal. Hb Renert, a slightly unstable variant, was found in a man from Cape Verde who also carried Hb S and presented with chronic hemolysis. The structural abnormalities were characterized by protein structure methods involving reversed phase high performance liquid chromatographic separations of globins and peptides, followed by mass spectrometry studies (electrospray, ion trap, tandem mass spectrom-etry).

We studied 46 unrelated sickle cell anemia patients from the western region of Colombia which has the largest Black population of the country. Twenty-three children and 23 adults were studied. The distribution of haplotypes in the children was 58% Bantu, 38% Benin, and 4% Senegal, and in the adults it was 59.4% Bantu, 35.1% Benin, and 5.5% Senegal (p = 0.920). All 92 chromosomes had typical African haplotypes, Bantu 55.5%, Benin 34.8%, Senegal, 4.3%, and Cameroon, 5.4%. Our results suggest a lack of differential survival among patients with sickle cell anemia and typical β-globin gene cluster haplotypes. They also agree closely with historical data that indicate that most African slaves brought to Colombia originated from Angola (Bantu population) and the Sao Thomo Island in the Bight of Benin (Central West Africa).

Hb Bushey, found in a Chinese baby and his father, is a new variant with a point mutation leading to the substitution Phe→tLeu at position β122. Hb Casablanca, found in a family from Morocco, is a further example of a hemoglobin variant that carries two abnormalities in the same chain; the first is identical to that of Hb Bushey and the second to that of Hb J-Antakya [β65 (E9)Lys→Met]. Structural abnormalities of both Hbs were determined by protein chemistry methods including electrospray and tandem mass spectrometry. Their stability and oxygen binding properties were found to be identical to those of Hb A. Various mechanisms that may lead to two point mutations in the same chain are reviewed briefly.

We here report two new, clinically silent, hemoglobin variants in which the structural modification disturbs the oxygen-linked chloride binding. Hb Antananarivo [α1(NA1)Val→Gly] was found during a systematic hematological study in a 24-year-old woman, who originates from Madagascar. Hb Barbizon [β144 (HC1)Lys→Met] was found in several members of a French family. The oxygen binding properties of Hb Barbizon were similar to those of Hb Antananarivo showing, in vitro, a decreased chloride effect as compared to Hb A. In Hb Barbizon, the replacement of lysine β 44 by a methionine residue decreased from 4 to 2 the excess positive charges in the central cavity, thus leading to a reduction of about half of the chloride effect. For Hb Antananarivo, the mechanism is unclear since there is no difference in the number of positive charges in the central cavity but alterations are likely at the aloc2 interface.

Oxygen equilibrium studies of purified Hb Camperdown [βl04(G6)Arg→Ser] have revealed an increased oxygen affinity at acid pH, while it is decreased for pH values above 7.4. This accounts for an almost 40% reduction in the alkaline Bohr effect. The effects of chloride and organophosphate effectors on the oxygen affinity of Hb Camperdown are inhibited by 40-50%. in chloridefree Hepes buffer, Hb Camperdown exhibits a lower oxygen affinity than normal Hb A. The present results confirm the important role of the positively charged residues lining the β1β2 interface in regulating the functional properties of hemoglobin.

A new case of Hb Providence was discovered in a French Caucasian family presenting a mild polycythemia. Structural and functional studies of the two abnormal fractions (Hb Providence Asn and Asp) have been performed. These confirm the abnormal characteristics of Hb Providence described previously. Red cells containing Hb Providence were fractionated with a Percoll Albumin density gradient. The respective amounts of the two components were determined in the youngest and oldest cells. We observed a slight increase of Hb Providence Asp from 34 to 37% during the life span of the erythrocytes which confirms that the deamidation of Hb Providence Asn to Hb Providence Asp is a fast process, already present and close to its maximum, in the reticulocytes. Both abnormal components are stable in the presence of isopropanol.