Hemoglobin variants

Hemoglobin variants are often given die geographical name of dieir origin. 

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. 

Some of the abnormal fetal hemoglobin variants are the product of an allele of the structural locus and others of an allele of the % structural locus The structural abnormality In all eight variants of Hb-A2 has been determined ... 

The techniques mentioned here provide a basic approach to the study of hemoglobinopathies since the Identification of many variants Is dependent upon their net charge. However, certain changes In the tertiary structure of a hemoglobin variant may also alter Its electrophoretic behavior. This Is well documented for many unstable variants such as for Hb-K51n which migrates more slowly than Hb-A but In which the neutral valyl residue In position 398 Is replaced by the neutral methlonyl residue. It should also be mentioned that differences In the level of heme oxidation will cause differences In the electrophoretic mobility. 

The detection of abnormal hemoglobins In cord blood samples Is usually made with electrophoretic procedures. Four types of hemoglobin variants can be present, namely y-chaln variants, 3-chaln variants, and Hb-Bart s or yif Indicating some form of a-chaln deficiency or a-thalassemla. 

Unstable hemoglobin variants are often found In persons with unexplained hemolytic anemias. Below are some guidelines for their detection. 

Detection of Variants With Altered Functional Properties. When substitutions In either a-or 3-chains Involve amino acid residues that participate In the contact with heme or the contact between chains, changes In functional properties can occur and the determination of the oxygen affinity of the blood sample or of an Isolated hemoglobin variant Is desirable. Oxygen affinity Is affected by temperature, pH, salt concentration, the level of 2,3-dlphosphoglycerate (2,3-DPG), and to a lesser extent by the concentration of the hemoglobin. The concentration of 2,3-DPG In blood changes rather rapidly after collection and a... 

Structural Analyses of Hemoglobin Variants It has become Impossible to characterize nearly any abnormal hemoglobin by Its electrophoretic and/or chromatographic mobility only This Is most strikingly demonstrated by the fact that over fifty different variants behave similar to Hb-S In electrophoresis Characterization, therefore, often requires detailed structural analyses or the demonstration of a property unique to a specific variant Some of the techniques used In these studies will be... 

A detailed study of the structure of a hemoglobin variant still requires at least 100 to 200 mg of the pure protein. The protein can best be Isolated by chromatographic techniques. 

The presence of Individual chains In a hemoglobin variant can also be demonstrated by electrophoresis at alkaline pH after the protein has been dissociated Into Its subunits through exposure to 6 M urea In the presence of 3-mercaptoethanol. The buffer is either a barbital buffer or a tris-EDTA-boric acid buffer, pH 8.0 - 8.6, and contains 6 M urea and 3-niercapto-ethanol. Dissociation of the hemoglobin Into subunits Is best accomplished In a mixture of 1 ml 10 g% Hb (or whole hemolysate), 4 ml 6 M urea barbital or tris-EDTA-boric acid buffer, and 1 to 1.5 ml 3-mercaptoethanol. After 30 minutes to 1 hour the sample Is subjected to cellulose acetate or starch gel electrophoresis. Each chain has a specific mobility and an alteration In electrophoretic mobility easily Identifies the abnormal chain. 

Figure 8.5 Effect of pH on protein mobility. Hemoglobin A (pi 7.1) and Hemoglobin C (pi 7.4) were electrophoresed in eight of the McLellan native, continuous buffer systems (Table 8.1). The diagram is drawn to scale. Migration is from top to bottom as shown by the vertical arrows. Bands marked A or C indicate the positions of the two hemoglobin variants in each gel representation. The polarities of the voltages applied to the electrophoresis cell are indicated by + and - signs above and below the vertical arrows. Run times are shown below the arrows. Note the polarity change between the gel at pH 7.4 and the one at pH 8.2. This reflects the pis of the two proteins (and was accomplished by reversing the leads of the electrophoresis cell at the power supply).

Hemoglobin Variants There are almost 500 naturally occurring variants of hemoglobin. Most are the result of a single amino acid substitution in a globin polypeptide chain. Some variants produce clinical illness, though not all variants have deleterious effects. A brief sample follows ... 

Relative mobility of common hemoglobin variants on Sepraphore III cellulose acetate... 

Know the base for hemoglobin variants and why some are harmless and some are not. 

The mutation for sickle hemoglobin occurred at least three times in Africa and once on the Indian subcontinent. The disease has spread from Africa to the Mediterranean, areas of Turkey, North and South America, the Caribbean, and the United Kingdom. The sickle hemoglobin combined with common hemoglobin variants in those areas (3-thalassemia in the... 

Wada, Y. (2002) Advanced analytical methods for hemoglobin variants. J. Chromatogr. B, 781, 291-301. 

Special applications of CIEF have focused on studies of glycoproteins, antibodies, and proteins in serum (e.g., hemoglobin variants [30,37,39,77-90], transferrin forms [21,31,36,80,82,90,91]), and cerebrospinal fluid [92], The gly-coforms of recombinant human tissue-type plasminogen activator [93-95] and... 

Carbonic anhydrase, a-lactalbumin, trypsin inhibitor, oval-bumn, conalbumin, hemoglobin variants Ribonuclease, insulin, a-lactalbumin Lysozyme, a-chymotrypsinogen, ribonuclease A, cytochrome c... 

The reversed-phase HPLC separation of the tryptic peptides of hemoglobin variants using ammonium acetate buffers, generally 10 mM at pH... 

In addition to the examples discussed above, these various reversed-phase HPLC mapping procedures have subsequently found numerous other advocates. Selected recent achievements include application to human hemoglobin variants 9a, 182, 185), the a- and j8-chains of rat hemoglobin 186), polypeptide hormones 9a, 99, 163), porcine C5a anaphylatoxin 187), Aplysia neuroactive polypeptide 188), ACTH/jS-lipotropin precursors 97, 99, 189, 190), oncoproteins 157), chick liver dihydrofolate reductase 191), limulin 192), ATP-citratelyase 193), spore-specific proteins 194), cAMP-dependent protein kinases (/95, 1%), interferons 197), complement components 9a, 198), aj-macroglobulin 198a), lectins 199), phycobiliproteins 200), bovine mitochondrial-F, ATPase 201), collagens, tubulins, and other structural proteins 9a, 202). 

An improved procedure for the preparation of stable anion exchangers on porous microparticulate silicas has been described by Regnier and co-workers 232), These and related supports have found application in the profiling of hemoglobin variants and for peptide separation 236-239). 

The synthesis of each hemoglobin polypeptide chain is under separate genetic control, Evidence to this effect has been obtained from studies of critical families with (combinations of) specific hemoglobin variants these data are extensively reviewed by Huehns and Shooter (H33). Thus, separate loci exist directing the synthesis of a, p, y, 8, e, and C chains. All data, including data from complete structural analyses (SIS), in-... 

Hemoglobin Variants with Sdbstitittions op a Residue Participating IN THE Contact with the Heme Group... 

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