STRUCTURAL ALTERATIONS OF HORSERADISH-PEROXIDASE IN THE PRESENCE OF LOW CONCENTRATIONS OF GUANIDINIUM CHLORIDE

The presence of very low concentrations of guanidinium chloride (GdmCl ) alters the tertiary structure of the monomeric heme-containing enzym e, horseradish peroxidase (HRP). The change in tertiary structure of t he protein was reflected in the mean fluorescence lifetime of its sing le tryptophan residue, which increased from 2.3 +/- 0.1 ns in the nati ve enzyme to 2.7 +/- 0.2 ns in the presence of 100 mM GdmCl. More conv incing evidence in support of such alterations came from quenching stu dy of tryptophan fluorescence using the most widely used quencher, acr ylamide. It revealed significant differences between the Stern-Volmer quenching constants observed in the absence and in the presence of Gdm Cl concentrations below 100 mM. The fluorescence emission maximum of 6 -propionyl-2-(dimethylamino)naphthalene (PRODAN), used as an extrinsic fluorophore to probe any changes in the tertiary structure of the enz yme, was blue-shifted from 522 nm in aqueous buffer to 509 nm in the p resence of 27 mu M native HRP. However, this emission maximum appeared at 519 nm when the PRODAN was incorporated in HRP previously incubate d with 100 mM GdmCl. The fluorescence lifetime of PRODAN incorporated in HRP was also different from that of PRODAN in buffer, but much more so in absence of GdmCl than in its presence. Taken together, these re sults indicate partial unfolding of HRP leading to a conformation with native-like secondary structure and unaltered enzymatic activity, in presence of millimolar concentrations of GdmCl.