How to Determine Hemoglobin Subunit Stoichiometry

Hemoglobin, the oxygen-carrying protein in red blood cells, is composed of two α and two β globin subunits. Understanding its quaternary structure is essential in biochemistry and clinical studies. To experimentally determine this stoichiometry, SDS-PAGE and gel filtration chromatography are two reliable and complementary techniques.

Correct Answer: 3. SDS-PAGE and gel filtration chromatography

Detailed Explanation:

1. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

• SDS-PAGE denatures proteins and separates them based on molecular weight.

• It helps identify individual polypeptide chains (e.g., α and β subunits of hemoglobin).

• Since α and β subunits have different molecular weights, they appear as distinct bands on the gel.

• This allows quantification of each subunit to verify that there are equal amounts of α and β.

2. Gel Filtration Chromatography (Size-Exclusion Chromatography)

• This technique separates native proteins based on their size and shape.

• Hemoglobin in its native tetrameric form (α₂β₂) elutes at a specific volume corresponding to its molecular size.

• This helps confirm the overall molecular weight and intact quaternary structure.

Why Not the Other Options?

• 1. SDS–PAGE and hydrophobic interaction chromatography: Hydrophobic interaction chromatography doesn’t provide stoichiometric information about subunits.

• 2. Gel filtration and ion-exchange chromatography: While gel filtration is helpful, ion-exchange chromatography is more suited for separation based on charge, not subunit analysis.

• 4. Isoelectric focusing and Native PAGE: These separate proteins based on pI and native size/charge, but are less accurate for precise stoichiometric determination of subunits.

Conclusion:

To determine the stoichiometry of α and β subunits in hemoglobin, researchers commonly use a combination of SDS-PAGE (for identifying and quantifying individual subunits) and gel filtration chromatography (for confirming the native tetrameric structure). These techniques together offer a complete picture of hemoglobin’s quaternary architecture.

Correct Answer: 3. SDS-PAGE and gel filtration chromato