Question

What observations would be expected to be seen when doing gel filtration with hemoglobin and myoglobin? Since these are both very large proteins, would they be expected to elute down the column at similar volumes?

Answer 1

Haemoglobin is a tetramer (4 subunits; ?₂?₂) of 16000 Dalton each. Therefore total molecular weight of haemoglobin is around 64,000 Dalton. Molecular weight of myoglobin is around 16,700 Dalton which is almost equal to a single subunit of haemoglobin. Both haemoglobin and myoglobin have almost similar structure and functions but haemoglobin is tetramer (forms quaternary protein structure) whereas myoglobin is monomer (tertiary protein structure). It means haemoglobin is larger as compared to myoglobin.

In Gel Filtration Chromatography heavy/ large molecules are eluted first followed by light/ small molecules depending on molecular weight/ size. Therefore during elution haemoglobin will be eluted first followed by myoglobin.