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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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The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium.

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The environmentally sensitive fluorescent probes 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) and 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanioc acid (DANCA) form complexes with the heme binding site of apohorseradish peroxidase. The dissociation constants of the PRODAN and DANCA complexes were determined from anisotropy titration data to be approximately 8.7 x 10(-5) and 3.3 x 10(-4) M, respectively. From comparison of the steady state fluorescence spectra of PRODAN and DANCA in solvents of varying dielectric constants, and in the apohorseradish peroxidase complex, we conclude that the heme binding site of horseradish peroxidase is relatively polar. The lifetimes of PRODAN and DANCA in organic solvents of varying polarities can be fit to single exponential decays. However, the lifetimes of PRODAN and DANCA associated with apohorseradish peroxidase, determined using a background subtraction method to correct for the non-negligible fluorescence of unbound probe, fit best to a distribution of lifetime values. We attribute these lifetime distributions to microenvironmental heterogeneity which is also consistent with the observed dependence of the emission maxima of PRODAN-apohorseradish peroxidase upon the excitation wavelength. In neither the PRODAN nor the DANCA case was evidence found in the time-resolved data for relaxation of the protein matrix around the excited state probe dipole.

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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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The local motion of protoporphyrin IX in the heme pocket of horseradish peroxidase has been studied using fluorescence methods. The temperature dependence of the anisotropy and lifetime of a protoporphyrin IX-apo-horseradish peroxidase complex, dissolved in a solution of 80% glycerol and 20% buffer (0.1 M phosphate, pH 7.4), was determined. Anisotropy data were analyzed in terms of the thermal coefficient of the frictional resistance to fluorophore movement. The resultant 'Y' plot was characterized by three distinct slopes. The slope corresponding to the lowest temperature regime agreed with the value obtained for fluorophores not complexed with protein. The slope corresponding to an intermediate temperature was lower indicating a larger resistance to porphyrin rotation. At higher temperatures this resistance to rotation diminished as evidenced by the increased slope. These results are contrasted with those obtained with the protoporphyrin IX-apomyoglobin complex.

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The quenching by molecular oxygen of the fluorescence of several probes complexed to apohorseradish peroxidase has been studied by intensity and time-resolved fluorescence methods. The probes utilized include 1-anilino-8-naphthalene sulfonic acid, 4,4'-bis (1-anilino-8-naphthalene sulfonic acid), and 2-p-toluidinylnaphthalene-6-sulfonic acid. These results are contrasted to those obtained using apohorseradish peroxidase complexed with protoporphyrin IX. The resistance of these complexes to denaturation by guanidine hydrochloride was determined. The results demonstrate a dramatic increase in oxygen accessibility to the naphthalene probes compared to protoporphyrin IX, which can be correlated to the increased stability of the protein-protoporphyrin IX complex.

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The quenching by molecular oxygen of the fluorescence from a protoporphyrin IX adduct of horseradish peroxidase has been investigated using both intensity and time-resolved techniques. The bimolecular quenching rate constant determined for this process, as evaluated by the conventional Stern-Volmer analysis, was 2 x 10(8) M-1 s-1, among the lowest observed for protein systems. This result suggests that the heme binding site in horseradish peroxidase is relatively inaccessible to oxygen, which may account for the observation of room temperature phosphorescence in aerated solutions from enzymatically created triplet states.

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The hemin moiety of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was removed and the apoprotein reconstituted with the fluorescent protoporphyrin IX. Steady-state and time-resolved fluorescence properties of the HRP(desFe) adduct were examined; the multifrequency phase and modulation method was utilized for lifetime and dynamic polarization studies. The emission spectrum of HRP(desFe) had maxima at 633 and 696 nm. The lifetime of this emission was characterized by a single exponential decay of 16.87 ns at 22 degrees C. Debye rotational relaxation times for HRP(desFe) were determined using both static (Perrin plot) and dynamic (differential phase and modulation fluorometry) methods; these two approaches gave values of 96 and 86 ns, respectively. A spherical protein of HRP's molecular weight and partial specific volume would be expected to have a Debye rotational relaxation time, at 22 degrees C, in the range of 50 to 60 ns, depending upon the extent of hydration. Hence our results indicate that HRP(desFe) is asymmetric; the global rotational relaxation times observed are consistent with those of a prolate ellipsoid with an axial ratio of 3:1.

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Conjugates of fluorescein isothiocyanate with clupein YI, YII and Z, the protamines from Clupea palasii, were prepared and their fluorescence utilized to determine the rotational relaxation times of the proteins. All conjugates exhibited single component lifetimes near 4.05 ns. Linear isothermal Perrin plots were obtained for all conjugates; these data indicated rotational relaxation times of 3.33 ns for clupein YI and YII and 3.19 ns for clupein Z. These results and the results from our previous studies lead us to postulate globular conformations for the three proteins with hydrated molecular diameters of 22 A. Based on these findings a three dimensional model for Clupein YII is proposed.

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