p> I have a question about problem 2-3, part E. The answer states that the best method of separating 2 polypeptides (one with no overall charge, the other with unknown charge, connected by hydrophobic forces, and with equal MW and shape) is gel electrophoresis w/o SDS. I was under the impression that all of the protein purification methods we discussed in class worked for the separation of proteins, not polypeptides that make up a single protein. Is this indeed the case? And if not, would you not have to denature this protein in order to break the hydrophobic connections between its 2 polypeptides?
Most of methods we discussed apply equally well to single polypeptide chain proteins or proteins that have quaternary structure (multi-polypeptide proteins). The exception is SDS-PAGE, since the denatures the proteins, most multi-polypeptide proteins would be converted to lower MW polypeptide chains. But I see the source of your concern: on the one hand you are told that the enzyme consists of 2 polypeptide chains, which would be held together by weak bonds. On the other hand, the answer says the best way to separate these 2 polypeptides would be native PAGE. Yet in native PAGE one would expect the quaternary structure to be maintained, since it is carried out under non-denaturing conditions. The resolution must be that you are given the 2 individual chains, already disassociated by an unnamed method, and now are asked to purify (separate) one from the other. Since they are the same MW and shape, one must rely on a possible charge difference, so native PAGE is called for.