H. Lam

Separation of tryptic peptides of normal and abnormal a, β, γ, and δ hemoglobin chains by high-performance liquid chromatography

Abstract High-performance liquid chromatography (HPLC) was used to separate tryptic peptides of the normal a , β, γ, and δ chains of human hemoglobins A, F, and A 2 and of the abnormal chains of 25 hemoglobin variants. In addition, the separation of chymotryptic peptides of he oxidized core of the normal a chain by HPLC was evaluated. HPLC has several advantages over conventional methods used for the separation of proteolytic fragments of hemoglobin chains. The method is fast, and reproducible, and requires only small quantities of material. Several peptides are eluted as single zones, thus eliminating the need of rechromatography for further purification. Characteristic changes in the elution pattern of the peptides often indicate specific modifications.

Hb Wuming or alpha 2 11(A9)Lys substituting for Gln beta 2.

A fast-moving hemoglobin variant was found in five members of a Chinese family of the Wuming district. The relative amount of this alpha chain variant in the heterozygote was about 20%. The abnormality caused no ill effects in its carriers. Sequence analysis identified a Lys substituting for Gln substitution at position alpha-11 (A9).

Hemoglobin Hofu or αβ [126 (H4) Val → Glu] Found in Combination with Hemoglobin S

Hb Hofu, αβ[126 (H4) Val → Glu], was found in 10 members of 2 apparently unrelated Valmiki families in central India. None showed evidence of hemolysis and hemoglobin levels were normal in most. In two individuals, Hb Hofu occurred in combination with Hb S, but neither had clinical manifestations of sickle cell disease. In samples containing Hb Hofu, the isopropanol precipitation test was positive. Quantitation of the hemoglobin fractions by DEAE-cellulose chromatography showed that Hb Hofu constituted a mean of 23-25% of the total whether in combination with Hb A or Hb S.

Heterozygosity and Homozygosity for the High Oxygen Affinity Hemoglobin Tarrant or α126 (H9) ASP→ASN in two Mexican Families

Two Mexican families from the State of Jalisco have been studied in which 11 members were carriers of Hb Tarrant. Ten subjects were Hb Tarrant heterozygotes producing about 25% of the abnormal hemoglobin. One 9-year-old boy was homozygous for Hb Tarrant. About 50% of his hemoglobin was of the variant type. The heterozygotes had mild erythrocytosis which was considerably more severe in the homozygote. The average P50 value for blood of the heterozygote was 15.1 mm Hg (controls: 22.5 mm Hg) while this value was decreased to 9 mm Hg in the homozygote. The clinical condition of the homozygote is compatible with a mild chronic tissue hypoxia.

Hb A2-Manzanares Or α2δ2 121(Gh4) Glu→Val, An Unstable δ Chain Variant Observed in a Spanish Family

A slowly moving variant of Hb A2 was present in a 25-year-old Spanish woman and her mother, and was found to have a Glu→Val substitution at position 121 of the δ chain. The variant was unstable and precipitated gradually during anion exchange chromatography. The effect of this substitution on the stability of the protein is apparently more severe for Hb A2 than for Hb A because Hb Beograd (β121 Glu→Val) has normal physicochemical properties.

HB A2-Canada Or A α2δ2 99(G1) ASP → ASN, a Newly Discovered Delta Chain Variant With Increased Oxygen Affinity Occurring In Cis to β-Thalassemia

An Indian family is described in which the father has a delta chain abnormal hemoglobin which is the result of a mutation of the delta gene in cis to a beta-thalassemia heterozygosity. The abnormality concerns a substitution of the Asp residue in position 99 (G1) by an Asn residue. A similar substitution has been found in the beta chain of Hb Kempsey (alpha 2 beta 2 99 Asp replaced by Asn). The observed abnormality results in a greatly increased oxygen affinity of this newly discovered Hb A2 variant.

Hb Nottingham (α;2β;2 (FG5) 98 VAL→GLY) in a Caucasian Male: Clinical and Biosynthetic Studies

A second instance of the unstable mutant Hb Nottingham (α;2β;2 (FG5) 98 Val→Gly) is reported in a 7-year-old boy. Because of splenomegaly, cholelithiasis, and frequent episodes of abdominal pain, he underwent a splenectomy and cholecystectomy at age 6. The surgery resulted in both an amelioration of his RBC destruction and an acceleration of his rate of growth.Biosynthetic studies were carried out using reticulocytes obtained from his peripheral blood. These analyses disclosed an exceedingly high specific activity ratio for Hb N/Hb A. In addition as the incubation proceeded, more radioactivity accumulated in the α; chain fraction than in the β; chains (β;A+β;N). This observation is presumed secondary to degradation of the unstable β;N chains.

Hb A2-Zagreb Or α2δ2125(H3)Gln→Lglu, A New δ Chain Variant in Association with δβ-Thalassemia

The structural identification of a new δ chain variant is described. The abnormal Hb A2 was found in two members of a family from Zagreb, Yugoslavia. The propositus also had a αβ-thalassemia heterozygosity.

Hb F-Kennestone or α2Gγ2 (EF1)77 His → Arg Observed in a Caucasian Baby

During our continuing attempts to characterize fetal hemoglobins (Hb F) with abnormal γ chains and abnormality was observed in a Caucasian female newborn which upon further analysis was found to be a His→Arg substitution at position 77 of the Cγ chain.

Hemoglobin G-San Jose (α2β27(A4)Glu←gly) in a Mexican Family

Hemoglobin G-San Jose was first discovered by Schwartz et al. in 1957 (1), and its structural abnormality was elucidated in 1959 (2,3). Since then, Ricco et al. (4) observed this variant in members of an Italian family while Musumeci et al. (quoted in 5) discovered a second Italian family with Hb G-San Jose. A blood sample from the propositus of this second family, an adult Sicilian man with Hb G-San Jose-β°-thalassemia, was used for an evaluation of some functional and physicochemical properties of the variant (5). These studies showed that the oxygen affinity and the Bohr effect of Hb G-San Jose, and its polymerization with Hb S were the same as those of Hb A. However, its mechanical precipitation rate was slightly increased while Hb G-San Jose was also slightly more heat labile.