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Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants. 1997

J Riou, and C Godart, and D Hurtrel, and M Mathis, and C Bimet, and J Bardakdjian-Michau, and C Préhu, and H Wajcman, and F Galactéros
Department of Biochemistry and INSERM U91, Hôpital Henri Mondor, Créteil, France.

A battery of relatively simple tests allows the presumptive identification of hemoglobin (Hb) variants, making unnecessary structural analysis by protein chemistry methods or DNA sequencing. The primary step in this strategy involves the use of a matrix of electrophoretic mobilities obtained under various experimental conditions. This leads to an unambiguous result in approximately 90% of the cases. Additional tests are required to characterize with more confidence the remaining 10%. We describe here the use of cation-exchange HPLC on the Bio-Rad Variant automated analyzer with the "beta Thalassemia Short" program. By comparing the elution time of 125 human Hb mutants, we found that some variants with almost identical pI values or produced by the same type of amino acid substitution displayed different elution times. We present several examples in which use of the HPLC profile helped establish the diagnosis.

UI MeSH Term Description Entries
D007525 Isoelectric Focusing Electrophoresis in which a pH gradient is established in a gel medium and proteins migrate until they reach the site (or focus) at which the pH is equal to their isoelectric point. Electrofocusing,Focusing, Isoelectric
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D002412 Cations Positively charged atoms, radicals or groups of atoms which travel to the cathode or negative pole during electrolysis. Cation
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D002852 Chromatography, Ion Exchange Separation technique in which the stationary phase consists of ion exchange resins. The resins contain loosely held small ions that easily exchange places with other small ions of like charge present in solutions washed over the resins. Chromatography, Ion-Exchange,Ion-Exchange Chromatography,Chromatographies, Ion Exchange,Chromatographies, Ion-Exchange,Ion Exchange Chromatographies,Ion Exchange Chromatography,Ion-Exchange Chromatographies
D005319 Fetal Hemoglobin The major component of hemoglobin in the fetus. This HEMOGLOBIN has two alpha and two gamma polypeptide subunits in comparison to normal adult hemoglobin, which has two alpha and two beta polypeptide subunits. Fetal hemoglobin concentrations can be elevated (usually above 0.5%) in children and adults affected by LEUKEMIA and several types of ANEMIA. Hemoglobin F,Hemoglobin, Fetal
D005914 Globins A superfamily of proteins containing the globin fold which is composed of 6-8 alpha helices arranged in a characterstic HEME enclosing structure. Globin
D006443 Hemoglobin A2 An adult hemoglobin component normally present in hemolysates from human erythrocytes in concentrations of about 3%. The hemoglobin is composed of two alpha chains and two delta chains. The percentage of HbA2 varies in some hematologic disorders, but is about double in beta-thalassemia. A2, Hemoglobin
D006444 Hemoglobin C A commonly occurring abnormal hemoglobin in which lysine replaces a glutamic acid residue at the sixth position of the beta chains. It results in reduced plasticity of erythrocytes.
D006446 Hemoglobin E An abnormal hemoglobin that results from the substitution of lysine for glutamic acid at position 26 of the beta chain. It is most frequently observed in southeast Asian populations.

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