Hemoglobin, isoelectric focusing

Manning JM et aJ Normal and abnormal protein subunit interactions in hemoglobins.] Biol Chem 1998 273 19359-Mario N, Baudin B, Giboudeau J Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing. J Chromatogr B Biomed Sci Appl 1998 706 123-Reed W, Vichinsky EP New considerations in the treatment of sickle cell disease. Annu Rev Med 1998 49 46l. 

Protein separation has commonly been achieved by isoelectric focusing (IEF). In various reports, IEF separations have been achieved with BSA [1036], bovine hemoglobin [1036], cytochrome c [189], P-lactoglobulin [189], protein markers [975,1037], and bacteria [1038],... 

Figure 8.8. Resolution by isoelectric focusing in an immobilized pH gradient (pH range 7.35-7.55 over 20 cm path) of two human fetal hemoglobins, one with glycine (lower) instead of alanine (upper) in position 136 of the gamma chains. The isoelectric points of the hemoglobins differ by only 0.003 pH units. (Courtesy of P. G. Righetti, University of Milano.)...

Dll. Drysdale, J. W., Righetti, P., and Bunn, H. F., The separation of human and animal hemoglobins by isoelectric focusing in polyacrylamide gel. Biochim. Bio-phys. Acta 229, 42-50 (1971). 

Capillary isoelectric focusing (CIEF) is a high-resolution technique for protein and peptide separation performed at academic sites and in the biotechnology and pharmaceutical industries for the analysis and characterization of, for example, recombinant antibodies and other recombinant proteins, isoforms of glycoproteins, point mutations in hemoglobin, and peptide mapping. Also, hyphenation to mass spectrometry and chip-based CIEF (microfabrication) have shown promise. CIEF kits and specific recipes/application notes are available from vendors of capillary electrophoresis (CE) equipment, as are a vast amount of publications and handbooks of CE published over recent years. 

Capillary isoelectric focusing coupled to mass spectrometry has gained popularity in recent years (by analogy to two-dimensional gel electrophoresis). Additional information obtained from mass spectrometry includes not only precise molecular-weight determination but also the possiblity for peptide sequencing. Analysis of hemoglobin variants, recombinant proteins, and monoclonal antibodies have been demonstrated [1,7,8]. 

Hempe JM, Vargas A, Graver RD. Clinical analysis of structural hemoglobin variants and Hb Ale by capillary isoelectric focusing. In Petersen JR, Mohammad AA, eds. Clinical and forensic applications of capillary electrophoresis. Totowa, NJ Humana Press, 2001 145-63. 

Mullins RE, Austin GE. Sensitivity of isoelectric focusing, ion exchange, and affinity chromatography to labile glycated hemoglobin. Chn Chem 1986 32 1460-3. 

Spicer KM, Allen RC, Buse MG. A simpfified assay of hemoglobin Ale in diabetic patients by use of isoelectric focusing and quantitative microdensitometry. Diabetes 1978 27 384-8. 

Two modifications of classical hemoglobin electrophoresis are also useful for studying these proteins isoelectric focusing and electrophoresis of individual globin chains, with or without the heme. These techniques can be used to identify some hemoglobins that are not separable by electrophoresis of the complete hemoglobin tetramers. 

Q. Tang, A.K. Harrata and C.S. Lee, High-resolution capillary isoelectric focusing-electrospray ionization mass spectrometry for hemoglobin variants analysis. Analytical Chemistry, 68, 2482-2487 (1996). 

Different proteins have been carbamylated to be used as charge standards. Carbamylated beta chains of hemoglobin (38) were reported to be stable standards, while Fawcett (39) took the approach of acetylation of myoglobin to produce charge standards for isoelectric focusing. Building on these standards, Tollaksen et al. 

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