Estimation of peptide/protein molecular weights by high-performance molecular-sieve chromatography on agarose columns in 6 M guanidine hydrochloride

Calibration curves showing a linear relationship between -log KD and (molecular weight)23 are presented for peptides/proteins subjected to molecular-sieve chromatography in 6 M guanidine hydrochloride on crosslinked gels of different agarose concentrations (9, 12 and 20%). These gels permit separation and estimation of molecular weights of peptides and proteins in the range 1000 and 90 000 daltons. Surprisingly, the exclusion limits in the presence of guanidine are only slightly affected by the gel concentration. Another unexpected result was that the exclusion limit in 6 M guanidine—HCl increased after crosslinking. In the absence of this agent a crosslinked gel has the same exclusion limit as the non-crosslinked one. Separations of model proteins are shown.