Globin chains analysis

In this analysis, the globin chains and heme in a red cell lysate are first dissociated using urea and dithiothreitol. The dissociated globin chains are then separated electrophoreti-caUy at both an alkaline and acid pH (Figure 31-15). HPLC may also be used for separation. Globin chains are identified by comparison of retention times and/or electrophoretic mobility with known reference materials or published data. Globin chain analysis is an adjunct to methods described earlier and in many laboratories has been discontinued and replaced with either HPLC or capillary isoelectric focusing. 

One or more abnormal a-globin chains can combine with Hb S. In African-Americans and West Africans the combination of Hb G Philadelphia (a68(E17) " > ) with Hb S is prevalent. HPLC analysis (Figure 31-12, i) on blood samples from these individuals shows at least two major peaks and two smaller peaks. The two major peaks are due to the combination of the normal a-chain with the normal P-chain and the abnormal a-chain with the normal P-chain. The two smaller peaks are due to the combination of the normal a-chain with the abnormal P-chain and the abnormal a-chain with the abnormal P-chain. Electrophoresis at alkaline pH shows major bands in the A and S positions with a minor band in the C position. At acid pH, bands are seen in the A and S positions. CBC analysis gives a slightly decreased Hb level with normal MCV and MCH. a- or p-thalassemia can be co-inherited with Hb G Philadelphia and Hb S. In these cases, CBC analysis results in markedly decreased MCV and MCH with reduced Hb concentration. 

Sugano M, Hidaka H, Yamauchi K, Nakabayashi T, Higuchic Y, Fujita K, et al. Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focussing method. Electrophoresis 2000 21 3016-9. 

Figure 3 Two-dimensional maps of globin chains (A) reduced but nonalkylated sample (B) reduced and alkylated sample prior to the focusing step. Note, in panel A, the presence of higher-order homooligomers of -chains, detected up to the dodecamer level. Note additionally the presence of homodimers of a-chains, as well as of a heterodimer a-fi. Note, in panel B, the complete removal of all higher-order oligomers and the detection of minor components of RBC lysates, such as carbonic anhydrase and thioreduction reductase, which would have been hidden under the string of dimers in nonalkylated samples. (Reproduced with permission from Herbert B, Galvan M, Hamdan M, et at. (2001) Reduction and alkylation of proteins in preparation of two-dimensional map analysis why, when and how Electrophoresis 22 2046-2057.)...

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