Structural perturbation of proteins in low denaturant concentrations

The presence of very low concentrations of the widely used chemical denatur ants, guanidinium chloride and urea, induce changes in the tertiary structu re of proteins. We have presented results on such changes in four structura lly unrelated proteins to show that such structural perturbations are commo n irrespective of their origin. Data representative of such structural chan ges are shown for the monomeric globular proteins such as horseradish perox idase (HRP) from a plant, human serum albumin (HSA) and prothrombin from ov ine blood serum, and for the membrane-associated, worm-like elongated prote in, spectrin, from ovine erythrocytes. Structural alterations in these prot eins were reflected in quenching studies of tryptophan fluorescence using t he widely used quencher acrylamide. stern-Vol mer quenching constants measu red in presence of the denaturants, even at concentrations below 100 mM, we re higher than those measured in absence of the denaturants. Both steady-st ate and time-resolved fluorescence emission properties of tryptophan and of the extrinsic probe PRODAN were used for monitoring conformational changes in the proteins in presence of different low concentrations of the denatur ants. These results are consistent with earlier studies from our laboratory indicating structural perturbations in proteins at the tertiary level, kee ping their native-like secondary structure and their biological activity mo re or less intact.