Juan E. Brunet

INTRAMOLECULAR TRYPTOPHAN HEME ENERGY TRANSFER IN HORSERADISH PEROXIDASE

Abstract— Chain folding of horseradish peroxidase allocates its sole tryptophanyl residue at a distance of 18 A from the active site heme group as determined by electronic energy transfer. This finding confirms that the phosphorescence spectrum observed in the peroxidase catalyzed oxidation of isobutyraldehydc is due to the excited triplet state acetone produced.

Apohorseradish peroxidase unfolding and refolding: intrinsic tryptophan fluorescence studies

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.

Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy

The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two‐photon Fluorescence Correlation Spectroscopy (FCS), and time‐resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5–1.5 M GdmCl, significant decreases in the steady‐state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two‐photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady‐state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems.

Singlet oxygen generation from the peroxidase-catalysed aerobic oxidation of an activated CH2 substrate☆

Abstract Photon emission, emission spectra, the product distribution, the effect of D2O and quenchers and steady state techniques were proposed to ascertain the involvement of singlet oxygen in a biological process. In particular, these experiments led to the conclusion that singlet oxygen is present in the reactions of malonaldehyde catalysed by horseradish peroxidase.

Time-resolved fluorescence studies on protoporphyrin IX-apohorseradish peroxidase

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 HRPs 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.

Dimethyl sulfoxide as chemical and biological probe: Conformational effect on peroxidase systems

Abstract The variation of the spectra and its reactivity towards 2-methylpropanal, indole-3-acetic acid and malonaldehyde of solutions of horseradish peroxidase in dimethyl sulfoxide-water mixtures has been studied. A broad pattern of changes was observed in the CD spectra of peroxidase, especially in the 400 nm region. These variations influenced strongly the excited triplet acetone emission from the 2-methylpropanal system which is generated in the active site of the enzyme protected from external quenching. This means that presumably the active site is more uncovered in the presence of dimethyl sulfoxide than the native form. Energy transfer parameters indicate that in fact there is a conformational effect produced by dimethyl sulfoxide in the horseradish peroxide active site. Dimethyl sulfoxide appears to be an important conformational probe in biochemistry.

OXYGEN DIFFUSION THROUGH HORSERADISH PEROXIDASE

Abstract— 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 × 108 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.

Fluorescence studies on clupein protamines: evidence for globular conformation.

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.