Reversed-phase chromatographic behavior of proteins in different unfolded states.

A series of standard small globular proteins in different unfolded states was studied by gradient reversed-phase liquid chromatography. The retention parameters Z [slope of log capacity factor (k') vs. log molar concentration of organic modifier, 1-propanol, in the mobile phase] and log I (the value of log k' at 1 M 1-propanol) were derived from gradient retention data. Each protein in four different conformational states, i.e., folded, chromatographic surface-unfolded, urea-unfolded and disulfide-bridge reduced-unfolded, showed a variation of 10-fold in Z and up to 10(12)-fold in I values. For the different states of all the proteins studied, the order of Z and I values was as follows: folded much less than surface-unfolded less than urea-unfolded less than reduced-unfolded. The differences in the values of the coefficients suggest, in agreement with literature reports, that proteins with their disulfide bridges cleaved have the largest degree of unfolding. In addition, the Z and I values and solution refolding kinetics all suggest that chromatographic surface-unfolded proteins have a lower degree of unfolding than their urea-unfolded forms. It was also found that an additional chemical cross-link in lysozyme caused a significant decrease in the first-order rate constant of the surface-induced unfolding process.