Structural Alterations of Horseradish Peroxidase in the Presence of low Concentrations of Guanidinium Chloride

Abstract

The presence of very low concentrations of guanidinium chloride (GdmCl) alters the tertiary structure of the monomeric heme-containing enz,yme, 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 f 0.1 ns in the native enzyme to 2.7 2 0.2 ns in the presence of 100 mM GdmCI. 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 SternVolmer 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 (PRO-DAN), 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 pM native HRP. However, this emission maximum appeared at 519 nm when the PRODAN was incorporated in HRP previously incubated with 100 mM GdmC1. 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 GdmCI.Keywords: horseradish peroxidase; guanidinium chloride; 6-propionyl-2-(dimethylamino)naphthalene; structural alterations ; fluorescence.Guanidinium chloride (GdmC1) has been very frequently used as a strong denaturant of proteins, which have been shown to undergo pronounced structural disintegrations at GdmCl concentrations above 1 M (Tanford, 1968(Tanford, , 1970. However, it is generally accepted that very low concentrations of GdmCl M) which are often present in the buffer during reconstitution of proteins from the completely unfolded state attained by treatment with 4-6 M GdmCI, do not impart any alterations in their tertiary structure. The present experiments are intended to analyze the effect of low concentrations of GdmCl on the tertiary structure of the monomeric enzyme, horseradish peroxidase (HRP) .HRP is a 44-kDa, monomeric heme-protein containing a single tryptophan residue at position 117, making it a good choice for fluorescence spectroscopic studies. The location of the Trp in the three-dimensional structure has not been idientified due to the absence of an X-ray crystallographic structure, but previous spectroscopic and chemical modification studies indicate that it is not a part of the catalytic site of the enzyme (Brunet et al., 1983;Pappa and Cass, 1993). A very low quantum yield of the tryptophan fluorescence is shwon to be due to intramolecular tryptophan-heme energy transfer in native HRP (Brunet et al., 1983). The denaturatiodrenaturation of both holo-HRP and apoCorrespondence to A. Chakrabarti, Biophysics D...

Binding of a Denatured Heme Protein and ATP to Erythroid Spectrin

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Interaction of the DNA‐binding antitumor antibiotics, chromomycin and mithramycin with erythroid spectrin

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