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 enzyme, horseradish peroxidase (HRP). The change in tertiary structure of the protein was reflected in the mean fluorescence lifetime of its single tryptophan residue, which increased from 2.3 +/- 0.1 ns in the native enzyme to 2.7 +/- 0.2 ns in the presence of 100 mM GdmCl. More convincing evidence in support of such alterations came from quenching study of tryptophan fluorescence using the most widely used quencher, acrylamide. It revealed significant differences between the Stern-Volmer quenching constants observed in the absence and in the presence of GdmCl 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 enzyme, was blue-shifted from 522 nm in aqueous buffer to 509 nm in the presence of 27 microM native HRP. However, this emission maximum appeared at 519 nm when the PRODAN was incorporated in HRP previously incubated 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 results 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.
Chakrabarti
A
A
Biophysics Division, Saha Institute of Nuclear Physics, Calcutta, India.
Basak
S
S
D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000737:chemistry*; D015394:Molecular Structure; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D006735:Horseradish Peroxidase; D019791:Guanidine