Because of similar electrophoretic mobilities, several hemoglobins cannot be differentiated on cellulose acetate medium. Electrophoresis of hemoglobins at pH 6.2 on agar gel may be useful in differentiating which hemoglobins?

• A. AI from A2
• B. A1 from D
• C. A, from E
• D. C from A2

Answer: (D)

Electrophoresis separates proteins by their net charge at the chosen pH, and the gel matrix can dramatically affect how far each protein moves. When two hemoglobin variants have very similar mobility on one medium, changing the pH and using a different gel type can reveal differences that were hidden before.

At pH 6.2 on an agar gel, the charges on the hemoglobins shift compared with the alkaline, cellulose-based system, and the matrix itself interacts with the proteins differently. Hb A2 is composed of alpha and delta chains, while Hb C is a variant of the beta chain with a specific amino acid change (which alters its charge). The delta chains confer a different overall charge and surface properties than the beta-chain variant present in Hb C, and the mutation in Hb C introduces a distinct charge that becomes evident under this acidic, gel-based condition. As a result, Hb C and Hb A2 migrate to different positions on agar at pH 6.2, allowing them to be distinguished from one another.