Oxygen diffusion near the heme binding site of horseradish peroxidase

Vargas, Victor Ignacio Cruz; Brunet, Juan E.; Jameson, David M.

doi:10.1016/0006-291x(91)91785-b

Cited by 6 publications

(9 citation statements)

References 20 publications

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“…The apoHRP conjugates with the naphthalene derivatives were prepared in phosphate buffer (0.1 M, pH 7.4) with at least a 20-fold molar excess of protein to rule out contribution from free probe. The stoichiometry of the protein-probe conjugates was measured by hemin titration following the heme absorption and the reduction of the probe fluorescence (Vargas et al, 1990). In all the conjugates a 1:1 stoichiometry was found.…”

Section: Methodsmentioning

confidence: 99%

Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes

Brunet

Vargas

Gratton

et al. 1994

Biophysical Journal

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Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered.

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Section: Methodsmentioning

confidence: 99%

Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes

Brunet

Vargas

Gratton

et al. 1994

Biophysical Journal

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“…Pappa and Cass also reported that the denaturation profile of apoHRP-PPIX, followed by tryptophan fluorescence, was very similar to that of apoHRP and concluded that PPIX in the active site does not stabilize the protein as well as hemin. Our previous results, however, demonstrated that PPIX does indeed stabilize the protein matrix as well as hemin (Vargas et al, 1991).…”

Section: Discussionmentioning

confidence: 68%

“…An issue related to discussions of apoHRP denaturation is the effect of hemin or PPIX on stabilization of the protein matrix. Our previous time-resolved fluorescence studies on apoHRP-PPIX (Jullian et al, 1989;Vargas et al, 1991;Brunet and Pulgar, 1993;Brunet et al, 1994) clearly demonstrated that the PPIX moiety is held very rigidly in the heme-binding site, which contradicts the assumption of Pappa and Cass (based on polarization data, which were not presented) that PPIX has "substantial mobility when bound to the protein" (Pappa and Cass, 1993). Pappa and Cass also reported that the denaturation profile of apoHRP-PPIX, followed by tryptophan fluorescence, was very similar to that of apoHRP and concluded that PPIX in the active site does not stabilize the protein as well as hemin.…”

Section: Discussionmentioning

confidence: 74%

“…2. The attribution of this intermediate state to a molten globule cannot be made with certainty, however, especially because the usual approach of studying ANS binding is precluded in this case, because ANS binds well to the heme-binding site region (Vargas et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…In the case of HRP, both intrinsic and extrinsic fluorescent probes have been utilized for this purpose. Extrinsic probes utilized include 1-anilino-8-naphthalene sulfonate (ANS), 4,4Ј-bis(1-anilino-8-naphthalene sulfonate (bis-ANS), 2-p-toluidiny-6-naphthalene sulfonate (TNS), and protoporphyrin IX (PPIX), which bind to and monitor the heme binding site (Ugarova et al, 1981;Jullian et al, 1989;Vargas et al, 1991;Brunet et al, 1994). HRP's single tryptophan residue (W117) also serves as an intrinsic probe that can monitor another region of the protein.…”

Section: Introductionmentioning

confidence: 99%

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Apohorseradish Peroxidase Unfolding and Refolding: Intrinsic Tryptophan Fluorescence Studies

Lasagna

Gratton

Jameson

et al. 1999

Biophysical Journal

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The unfolding and refolding of apohorseradish peroxidase, as a function of guanidinium chloride concentration, were monitored by the intrinsic fluorescence intensity, polarization, and lifetime of the single tryptophan residue. The unfolding was reversible and characterized by at least three distinct stages-the intensity and lifetime data, for example, were both characterized by an initial increase followed by a decrease and then a plateau region. The lifetime data, in the absence and presence of guanidinium chloride, were heterogeneous and fit best to a model consisting of a major Gaussian distribution component and a minor, short discrete component. The observed increase in intensity in the initial stage of the unfolding process is attributed to the conversion of this short component into the longer, distributed component as the guanidinium chloride concentration increases. Our results clarify and amplify previous studies on the unfolding of apohorseradish peroxidase by guanidinium chloride.

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Heme-Protein Fluorescence

Hirsch

Topics in Fluorescence Spectroscopy

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Oxygen diffusion near the heme binding site of horseradish peroxidase

Cited by 6 publications

References 20 publications

Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes

Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes

Apohorseradish Peroxidase Unfolding and Refolding: Intrinsic Tryptophan Fluorescence Studies

Heme-Protein Fluorescence

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