Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focusing method

To analyze both hemoglobin (Hb) and globin chain variants, we modified a co mmonly used method, capillary isoelectric focusing (CIEF), with detection a t 280 nm. The samples were hemolysates prepared from red blood cells, and g lobin chains obtained from the hemolysates by treatment with cold acidified acetone. When the migration time for the internal reference, carbonic anhy drase I (isoelectric point, pI 6.60), was taken as 1.0, the migration ratio for Hb A(0) in normal human blood was 0.877 +/- 0.004 (mean +/- SD, n = 9) , and those of the alpha- and beta-globin chains were 0.673 +/- 0.004 and 0 .847 +/- 0.005 (mean +/- SD, n = 4), respectively. The ratio of peak height s between the beta- and alpha-globin chains (beta/alpha) in the normal Hbs obtained from four subjects was almost constant at 2.5 +/- 0.1 (mean +/- SD ). This ratio indicates which of the globin chains includes a mutation (if one exists). When an Hb variant, Hb Hoshida (in which Gln is substituted fo r Glu at residue 43 in the beta-globin chain), was analyzed by this method, two main peaks were observed (migration ratios 0.836 and 0.877, correspond ing to an abnormal and the normal Hb, respectively). An additional peak wit h an abnormal migration ratio of 0.788 was also detected in the globin chai n profiles. The ratio of peak heights between normal beta- and alpha-globin chains was 1.57, indicating that a mutation exists in the beta-globin chai n. We thus established a convenient system using CIEF that provides a rapid and reproducible method for the random analysis of both Hb and globin chai n variants.