About the Difficulties with Stoichiometry Determinations

The MW of the subunits ascertained by SDS gel electrophoresis easily deviates from the real one by about 10 to 40%, because glycosylation, unusual amino acid composition, phosphorylation, sulfation, and so on let proteins run atypically. After covalent cross-linking of all subunits, SDS gel electrophoresis also delivers an estimate of the oligomer s MW. This estimate is even more uncertain than that for the individual subunits, because additional cross-linking molecules increase the MW, and cross-linking of the polypeptide chains sometimes changes their run speed in SDS gels and with it the apparent MW. 

A precise determination of the MW of the subunits is only possible by means of the MALDI-TOF mass spectrometer (see Section 7.5). However, as long as the MW of the oligomer cannot be determined with similar precision, number and stoichiometry of subunits (N S) remain uncertain and should be confirmed with other methods. There are three classical strategies for determining the subunit composition of an oligomer X-ray structural analysis, hybridization. 

The most precise method of determining the N S of a protein is X-ray structural analysis. A prerequisite for this technique is good crystals of the protein. Their production is difficult even for soluble proteins. With rare membrane proteins, it is almost impossible. Furthermore, the X-ray structural analysis is the domain of a few specialists and requires detailed knowledge of physics. 

Deisenhofer, J., et al. (1985). Stracture of the Protein Subunits in the Photosynthetic Reaction Center of Rho-dopseudomonas viridis at 3 A Resolution, Nature 318 618-624. 

These combinatorics can also be applied to heterooligomers and yield similar equations. In the 1960s, hybridization experiments with enzymes were fashionable. You either produced a from a or you isolated an isoenzyme, whose monomers a differed from a in their charge and nevertheless hybridized with a into an oligomer. For the hybridization, the oligomers were mixed in a certain proportion, split into subunits, and afterward associated into oligomers again. With native gel electrophoresis or lEC, the experimenter determined the number of the hybrids and with it the number of subunits. 

---