T. H. J. Huisman

Studies on the heterogeneity of hemoglobin : IX. The use of tris(hydroxymethyl)aminomethane—HCl buffers in the anion-exchange chromatography of hemoglobins

Abstract A modified procedure for the separation of various hemoglobin types by anion exchange chromatography has been presented. DEAE-Sephadex, A-50 medium, was preferred over DEAE-cellulose as chromatographic medium. Complete separations of many hemoglobin fractions were obtained by applying a single pH gradient to the columns using TRIS—HCl buffersof reasonably high concentrations (0.05 M). The method was applicable both for analytical and preparative purposes.

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.

Separation of Human Hemoglobins by Deae-Cellulose Chromatography using Glycine-Kcn-Nacl Developers

This chromatographic procedure uses DEAE-cellulose as ion exchanger and glycine-KCN-NaC1 solutions as developers. Blood samples from several adults and newborn infants with alpha, beta, delta, or gamma chains variants have been analysed. The hemoglobins are eluted as compact and symmetrical zones, and the separation of many hemoglobin types is greatly improved. The procedure is relatively fast, simple, and inexpensive.

Studies on the heterogeneity of hemoglobin : XIII. Chromatography of various human and animal hemoglobin types on deae-sephadex

Abstract A modification of the anion exchange chromatography of various hemoglobin types using DEAE-Sephadex is presented. It has been observed that the replacement of the DEAE-Sephadex A-50, 100 to 270 mesh, by a similar preparation, but particle size 40 to 120 μ, greatly improved the resolution of several human and animal hemoglobin types.

Separation of normal and abnormal hemoglobin chains by reversed-phase high-performance liquid chromatography

A review is presented of the elution patterns on reversed-phase columns of the normal and abnormal globin chains of different hemoglobin types, including 16 beta-chain variants, 7 alpha-chain variants, 9 gamma-chain variants, and 4 variants with fusion or hybrid chains. Separations appear to be based primarily on differences in hydrophobicity. The method is ideally suited for the detection of abnormal globin chains, their quantitation and their isolation. Semi-quantitative data based on the calculation of the delta/non-alpha ratios allow the detection of beta-thalassemic conditions in situations where the quantitation of hemoglobin A2 by other procedures is impossible or complicated.

THE PRESENT STATUS OF THE HETEROGENEITY OF FETAL HEMOGLOBIN IN β‐THALASSEMIA: AN ATTEMPT TO UNIFY SOME OBSERVATIONS IN THALASSEMIA AND RELATED CONDITIONS*

t Laboratory o f Protein Chemistry, Department of Cell and Molecular Biology, Medical College of Georgia and Veterans Administration Hospital Augusta, Georgia 30902

Fetal hemoglobin in normal adults and β-thalassemia heterozygotes

Division of Chetnistry and Chemical Engineering,

Molecular Characterization of α-Thalassemia Determinants, β-Thalassemia Alleles, and βs Haplotypes among Kuwaiti Arabs

California Institute of Technology Pasadena, California 91 109 5 Department of Physiology and Biochemistry, Faculty o f Agriculture Department of Pediatrics, Faculty of Medicine University of Skopje Skopje, Yugoslavia Childrens Hospital of Los Angeles and Department of Pediatrics School of Medicine, University of Southern California Los Angeles. California 90027 * * Department of Hematology, Hadassah Medical School Hadassah University Hospital, Hebrew University Jerusalem, Israel

Molecular characterization of β-globin gene mutations in Malay patients with Hb E-β-thalassaemia and thalassaemia major

SummaryA recently developed high performance liquid chromatographic (HPLC) procedure using a weak cation exchanger (PolyCAT) in columns of different sizes was used to quantify fetal hemoglobin (Hb F) in blood of normal adults and β-thalassemia (β-thal) heterozygotes with ten different types of mutations. Preparative PolyCATHPLC greatly facilitated the characterization of isolated Hb F, i.e., the determination of the relative quantities of the Gγ and Aγ chains. The method is accurate and allows quantitation of Hb F at the 0.5% level; preparative PolyCAT-HPLC allows isolation of (nearly) pure Hb F from blood samples with low (< 1%) Hb F. Adult Hb F levels were determined in 69 normal adults (including 24 diabetics); Hb F levels fell below 1% except for subjects with abnormal -Gγ-Gγ arrangement and a C→T mutation at position -158 relative to the Cap site of both Gγ genes. The effect of the same mutation in the normal-Gγ-Aγ-arrangement was variable. Certain β-thal mutations (namely, those at positions -29;-88; IVS-I-1; IVS-II-1) were associated with high Hb F levels in heterozygotes, while those at nucleotide (nt) positions IVS-I-6; IVS-I-110; codon 24; codon 39; codons 41/42; IVS-II-745 were not. Gγ values varied and often fell into two groups (high Gγ and low Gγ); high Gγ values were not associated with high Hb F values. The chromatographic procedure is ideally suited for Hb A2 quantitation. Average values of Hb A2 in β-thal heterozygotes with any one of nine of the ten mutations were twice that of normals; the one exception was the β-thal heterozygote with the IVS-I-6 (T→C) mutation with an average low Hb A2 value of 3.6%.