Question

4. The molecular weight of an Dalton, as determined by sedimentation equilibrium measurements and by gel filtration chromatography. The SDS-polyacrylamide gel electrophoresis (SDS PAGE) of the protein yields a single band corresponding to molecular weight of 70,000 Dalton. However, in the presence of the reducing agent, B-mercaptoethanol, the SDS PAGE shows two bands, corresponding to molecular weights of 30,000 and 20,000 Dalton. unspecified protein, at physiological conditions, is 70,000 ure a. From these data, describe the native protein in terms of the number of subunits present, their molecular weight, stoichiometry of subunits, and the kinds of bonding (covalent, noncovalent) existing between the subunits. (8 Pts) AYOAence

Answer 1

The native protein is a hetero dimer( protein having two different peptides as subunits) of 50.000 Da. One subunits weigh 30,000 Da and the other one weigh 20,000 Da. These subunits are bound by inter molecular disulfide (S-S) bonds- a covalent bond formed between two thiol groups of two aminoacid residues one from each subunit. That is why the protein weighed more in SDS PAGE , since disulfide bridges makes protein structure more convoluted that it slows down the protein's movement in PAGE, This makes the protein appear to have higher moelcular weight than it actually has.The reducing agent beta-mercaptothanol. breaks disulfide bonds and separate the protein's subunits.