Hemoglobins column chromatography

The column chromatography of the five common phenotypes were also studied by Hopkinson and Harris (H12). A 10-ml aliquot of the supernatant from a centrifuged hemolysate was applied to the DEAE column which had been washed with Tris-phosphate buffer (pH 8.0). The column was then washed with the starting buffer to elute the hemoglobin. The enzyme was eluted with an exponential gradient of sodium chloride in Tris-phosphate buffer and collected in 2-ml fractions at a flow rate of 20 ml/hour. 

Fig. 15. Horizontal starch gel electrophoresis to show the small differences in mobility between three fetal hemoglobin variants. Left to right (1) Hb-F-Malta-I heterozygote, (2) and (3) normal adults (4) Hb-Fx in a Negro newborn (5) Hb-F-Malta-II heterozygote (6), (7), and (8), Hb-A, Hb-F, and Hb-F-Malta-II fractions, respectively, isolated by column chromatography on CM-cellulose. Tris-EDTA-boric acid buffer, pH 9.0, o-dianisidine stain.

Figure 4 Total ion chromatograms for (A) normal cell hemoglobin (HbAo) and (B) sickle cell hemoglobin (HbS) produced from a five-column chromatography mass spectrometric analysis system. The peak numbers correspond to the peptide sequences listed in the upper left of A. Peptides identified that correspond to expected tryptic peptides are labeled T, with the number corresponding to the sequential position of the tryptic peptide in the protein sequence. The x and [i refer to the a and /i chains of hemoglobin. The oniy peak differing in retention time and mass of the observed peptide is peak 4 in chromatogram B. (From Ref. 66.)...

If, however, the reticulocytes were incubated without added iron or hemin (control), and the hemoglobin was first isolated from the ribosome-free hemolysate by column chromatography on carboxymethyl-cellulose or Sephadex G 100, the a/p ratio was much less than one (Tables 3 and 4). The addition of hemin to the incubation medium resulted in an increase in the specific activity of the globin and of the... 

Chromatography of hemoglobins on columns of DEAE-Cellulose (DE-52 mlcrogranular, preswollen Whatman) often results In excellent separations of many variants, because the hemoglobins are eluted as sharp, narrow zones generally widely separated from each other The hemoglobin zones are eluted from the DE-52 columns at a distinctly higher pH value than from a similar DEAE-Sephadex column ... 

Fig. 6. Separation of minor hemoglobin components by chromatography on columns of CM-Sephadex. (I) A freshly prepared hemolysate. (.2) Same hemolysate, aged for 40 days. The values in parentheses represent the relative amounts of the individual zones the elution pH value of each individual zone is given at the top of the first chromatogram.

Fig. 21. Separation of hemoglobin components by chromatography on a column of CM-cellulose. Red cell hemolysates from a 19-year-old female patient with sickle cell anemia (top graph), and from a subject with a heterozygosity for the a chain variant Hb-St. Luke s (bottom graph).

Ampholyte displacement chromatography [43] does not require special packings to be applied. Conventional ion exchangers are sufficient. The main principle is the elution of the column with carrier ampholytes. Leaback and Robinson [43] used CM-cellulose for separation of acetyl hexosamidase isoenzymes (Fig. 4.5.3), Young and Webb [44,50] separated serum proteins on DEAE-cellulose, and Chapuis-Cellier et al. [97] described ampholyte displacement chromatography of hemoglobins on DEAE-cellulose. 

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