UNIVERSAL CALIBRATION OF SIZE-EXCLUSION CHROMATOGRAPHY FOR PROTEINS IN GUANIDINIUM HYDROCHLORIDE INCLUDING THE HIGH-MOLECULAR-MASS PROTEINSTITIN AND NEBULIN

The chromatography of polypeptides in 6 M guanidinium hydrochloride (G uHCl) was studied with respect to the measurement of two ultra-large p olypeptides, titin and nebulin. These proteins are integral constituen ts of the muscle structure, responsible for elasticity, and play an im portant role in muscle function. Both are outside the previously avail able range of calibration standards. This problem was circumvented by universal calibration in two different solvents, namely denaturing GuH Cl conditions for the unknown polypeptides and buffered solutions of v iruses under native assembly conditions. The accuracy of the approach was established. Two matrices were tested for their stability towards this solvent change without changing their calibration graphs. For Sup erose-6 the calibration changed by 10%. TSK-6000PW exhibited a congrue nt calibration graph for native and denaturing conditions. By extrapol ation it was possible to estimate the chain molecular masses for nebul in to be 560 000 and that for T-II, the extractable form of titin, to be 2 000 000.