Hemoglobin S can be separated from hemoglobin D by which method?

Hemoglobin S can be separated from hemoglobin D by which method?

• A. agar gel electrophoresis at pH 5.9
• B. thin layer chromatography
• C. alkali denaturation
• D. ammonium precipitation

Answer: (A)

The question tests how hemoglobins are separated by charge at a fixed pH. In electrophoresis, proteins migrate according to their net electric charge, so amino acid substitutions that alter charge change how far they move. At an acid pH of about 5.9, HbS (beta-globin mutation Glu to Val) and HbD (beta-globin mutation Glu to Gln) acquire different net charges, causing them to travel to distinct positions on an agarose gel. This results in separate bands for HbS and HbD, allowing you to distinguish the two variants clearly. The other methods don’t provide that distinct separation pattern. Thin-layer chromatography relies on polarity and interaction with the stationary phase, which isn’t tuned to reliably separate these two specific variants. Alkali denaturation is a screening test for the presence of HbS but doesn’t separate HbS from HbD. Ammonium precipitation is based on solubility differences and likewise doesn’t resolve these two variants into separate, identifiable bands.

The question tests how hemoglobins are separated by charge at a fixed pH. In electrophoresis, proteins migrate according to their net electric charge, so amino acid substitutions that alter charge change how far they move. At an acid pH of about 5.9, HbS (beta-globin mutation Glu to Val) and HbD (beta-globin mutation Glu to Gln) acquire different net charges, causing them to travel to distinct positions on an agarose gel. This results in separate bands for HbS and HbD, allowing you to distinguish the two variants clearly.

The other methods don’t provide that distinct separation pattern. Thin-layer chromatography relies on polarity and interaction with the stationary phase, which isn’t tuned to reliably separate these two specific variants. Alkali denaturation is a screening test for the presence of HbS but doesn’t separate HbS from HbD. Ammonium precipitation is based on solubility differences and likewise doesn’t resolve these two variants into separate, identifiable bands.