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J W Petrich

Publications and source records attributed to J W Petrich.

At least 19 recordsLinked to original sources
PubMed2001

Steady-state and time-resolved spectroscopy of F420 extracted from methanogen cells and its utility as a marker for fecal contamination.

Methanogenic bacteria, which are common inhabitants of the animal digestive tract, contain the fluorescent compound F420 (coenzyme 420), a 7,8-didemethyl-8-hydroxy-5-deazariboflavin chromophore. F420 was characterized as an initial step in determining if this compound would be useful as a fluorescent marker for the detection of fecal and ingesta contamination. Using a single anion exchange chromatographic process, F420 was separated from other cell components of a Methanobrevibacter sp. cell culture. The extent of separation was determined spectroscopically. To aid in the development of possible techniques for the detection of fecal contamination using F420 as a marker, further spectroscopic investigation of F420 was conducted using steady-state and time-resolved fluorescence methods. The fluorescence lifetime of F420 in an elution buffer of pH 7.5 was found to be 4.2 ns. At higher pH values, the fluorescence decay, F(t), was best described by a sum of two exponentials: at pH 13, F(t) = 0.31 exp(-t/4.20 ns) + 0.69 exp(-t/1.79 ns). Further investigation using front-faced fluorescence techniques has shown that emission from F420 can be collected efficiently from samples of methanogen cell cultures as well as from fecal material.

Animals↗
PubMed2001

Is the excited-state H-atom transfer in hypericin concerted?

The excited-state intramolecular H-atom transfer of hypericin (Hyp) was investigated as a function of pH in monodispersed reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane/water and in complexes with Tb3+ under conditions in which one of the two carbonyl groups of Hyp is incapable of accepting a hydrogen atom. The results of pump-probe transient absorption experiments provide no evidence for a concerted H-atom transfer mechanism.

Anthracenes↗
PubMed2000

Effect of pH on the fluorescence and absorption spectra of hypericin in reverse micelles.

The well-characterized, monodispersed nature of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane and their usefulness in approximating a membrane-like environment have been exploited to investigate the effect of pH and water pool size on the photophysical properties of hypericin (Hyp). Our measurements reveal two titratable groups of pKa approximately 1.5 and approximately 12.5. These are assigned to the HypH+/Hyp equilibrium (the deprotonation of a carbonyl group) and the Hyp-/Hyp2- equilibrium (the deprotonation of a peri hydroxyl group). The low-energy absorbance maxima of HypH+, of Hyp and Hyp- and of Hyp2- are 583, 594 and 613 nm, respectively. Neither at pH 13 nor at 1 M HCl is the system entirely in the Hyp2- or the HypH+ forms. Ours is the first study of Hyp in reverse micelles as well as the first time-resolved study of Hyp as a function of pH.

Anthracenes↗
PubMed2000

Multidimensional reaction coordinate for the excited-state H-atom transfer in perylene quinones: importance of the 7-membered ring in hypocrellins A and B.

The excited-state intramolecular H-atom transfer reactions of hypocrellins B and A are compared by using time-resolved absorption and fluorescence upconversion techniques. The hypocrellin B photophysics are well described by a simple model involving one ground-state species and excited-state forward and reverse H-atom transfer with a nonfluorescent excited state. We suggest that excited-state conformational changes are coupled to the H-atom transfer in hypocrellin B just as gauche/anti changes are coupled to the H-atom transfer in hypocrellin A.

Kinetics↗
PubMed1999

Subcellular distributions and excited-state processes of hypericin in neurons.

The photodynamic drug, hypericin, is studied in fetal rat neurons using fluorescence microscopy. Hypericin has an extremely high affinity for the cell membrane and is found to a smaller extent in the nucleus. Fluorescent excitation of hypericin is shown to cause irreversible damage to the cell membranes of living neurons. Fixed cells were used to make ultrafast time-resolved measurements to avoid the deleterious effects of long-term exposure to intense light and room temperatures. To our knowledge, these are the first ultrafast time-resolved measurements of the fluorescence lifetime of hypericin in a subcellular environment. Nonexponential fluorescence decay is observed in hypericin in the neurons. This nonexponential decay is discussed in terms of other examples where nonexponential decay is induced in hypericin upon its binding to biomolecules. The nonradiative processes giving rise to the nonexponential hypericin decay are attributed to excited-state electron transfer, excited-state proton transfer or both.

Animals↗
PubMed1999

Photophysics of hypericin and hypocrellin A in complex with subcellular components: interactions with human serum albumin.

Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and approximately 5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbonyl oxygen and the N1-H of the tryptophan residue in the IIA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of approximately 3 x 10(-15) s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the approximately 5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.

Anthracenes↗
PubMed1999

Fluorescence properties of recombinant tropomyosin containing tryptophan, 5-hydroxytryptophan and 7-azatryptophan.

Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptophan (5OH122W) or 7-azatryptophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent-exposed position c of the coiled-coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue-shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2-3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple-exponential fluorescence decay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7-azatryptophan. The range of lifetimes observed for 5-hydroxyindole and 5-hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 5OH122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 5OH122W.

5-Hydroxytryptophan↗
PubMed1998

Domain structure of the Staphylococcus aureus collagen adhesin.

Sequence analysis of surface proteins from Gram-positive bacteria indicates a composite organization consisting of unique and repeated segments. Thus, these proteins may contain discrete domains that could fold independently. In this paper, we have used a panel of biophysical methods, including gel permeation chromatography, analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy, to analyze the structural organization of the Staphylococcus aureus collagen adhesin, CNA. Our results indicate that the structure, function, and folding of the ligand-binding domain (A) are not affected by the presence or absence of the other major domain (B). In addition, little or no interaction is observed between the nearly identical repeat units within the B domain. We propose that CNA is indeed a mosaic protein in which the different domains previously indicated by sequence analysis operate independently.

Adhesins, Bacterial↗
PubMed1998

Hypocrellin A photosensitization involves an intracellular pH decrease in 3T3 cells.

The fluorescent pH probe carboxy-seminaphtorhodafluor-1 (C-Snarf-1) has been used for laser microspectro-fluorometric assays of intracellular pH in 3T3 mouse fibroblasts treated with hypocrellin A. These results are compared to those previously obtained with the structurally related hydroxylated polycyclic quinone, hypericin (Sureau et al., J. Am. Chem. Soc. 118, 9484-9487, 1996). A mean local intracellular pH drop of 0.6 units has been observed in the presence of 1 microM hypocrellin A after 90 s of exposure to 0.1 microW of laser irradiation at 514.5 nm. The time evolution of the cytoplasm acidification for hypocrellin A-treated cells is faster than that for cells treated by hypericin. Thus, release of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin. In addition, the pH dependence of the quenching of C-Snarf-1 fluorescence in 3T3 cells under continuous irradiation has been observed. It is shown here that under continuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C-Snarf-1 that results in an increase of C-Snarf-1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hypericin or its analogs may be involved in the production of other photoreactive species. Finally, we suggest that, just as for hypericin, this pH drop may be involved in the antiviral and antitumor activity of hypocrellin A.

3T3 Cells↗
PubMed1998

The role of oxygen in the antiviral activity of hypericin and hypocrellin.

The light-induced antiviral activity of hypericin and hypocrellin in the presence and absence of oxygen was examined under experimental conditions where the effect of oxygen depletion could be quantified. There was a significant reduction of light-induced antiviral activity of hypericin and hypocrellin under hypoxic conditions. Interestingly, antiviral activity of hypocrellin was not observed at low oxygen levels at which hypericin retained measurable virucidal activity. This suggests that additional pathways, such as the generation of protons from excited states of hypericin, may enhance the biological activity of activated oxygen species.

Adenosine Triphosphate↗
PubMed1995

Roles of oxygen and photoinduced acidification in the light-dependent antiviral activity of hypocrellin A.

Hypocrellin A displays photoinduced antiviral activity, in particular against the human immunodeficiency virus (HIV), as does its counterpart, hypericin. Although hypocrellin A, like hypericin, executes an excited-state intramolecular proton transfer, it differs from hypericin in two important ways. Unlike hypericin, hypocrellin A absolutely requires oxygen for its antiviral activity. Also, whereas we have previously demonstrated that hypericin functions as a light-induced proton source, we do not observe that hypocrellin A acidifies its surrounding medium in the presence of light. These results are discussed in the context of the ground- and excited-state photophysics of hypericin and its mechanisms of photoinduced virucidal activity.

Anthracenes↗
PubMed1994

Chemiluminescent activation of the antiviral activity of hypericin: a molecular flashlight.

Hypericin is a naturally occurring photosensitizer that displays potent antiviral activity in the presence of light. The absence of light in many regions of the body may preclude the use of hypericin and other photosensitizers as therapeutic compounds for the treatment of viral infections in vivo. The chemiluminescent oxidation of luciferin by the luciferase from the North American firefly Photinus pyralis was found to generate sufficiently intense and long-lived emission to induce antiviral activity of hypericin. Light-induced virucidal activity of hypericin was demonstrated against equine infectious anemia virus, a lentivirus structurally, genetically, and antigenically related to the human immunodeficiency virus. The implications for exploiting chemiluminescence as a "molecular flashlight" for effecting photodynamic therapy against virus-infected cells and tumor cells are discussed.

Animals↗
PubMed1994

Ultrafast measurements of geminate recombination of NO with site-specific mutants of human myoglobin.

Flash photolysis studies of NO recombination to heme proteins offer a direct probe of protein structural changes on the tens of picoseconds timescale where they can be compared with molecular dynamics simulations. The geminate recombination of NO to site-specific mutants of human myoglobin (Mb) was studied following photodissociation of the MbNO form. Single amino acid changes were introduced at positions Val68, His64, Lys45 and Asp60 because motions of residues at these positions are generally regarded as important for the mechanism of ligand binding. In sharp contrast to the properties of simple porphyrin-NO complexes, the rebinding kinetics are found to be non-exponential for all mutants, even in aqueous solution at 298 K. The Val68 and His64 mutants substantially affect the NO rebinding rates but, surprisingly, so do changes on the protein surface that are further away from the iron. These changes in kinetics occur on a tens of picoseconds timescale, and therefore there is either a fast communication between protein residues over quite long distances or there are subtle differences in protein structure that exert great control over the reaction dynamics. Various models for the rebinding kinetics are evaluated. A model-free approach to data analysis using the maximum entropy method is found to be most useful. This analysis shows that the rate distributions are very different for the mutants, but are generally bimodal.

Asparagine↗
PubMed1994

Synthesis and spectral characterization of 5'-phosphopyridoxyl-D,L-7-azatryptophan, a photophysical probe of protein structure and dynamics.

We report the first isolation of a unique adduct of pyridoxal 5'-phosphate, 5'-phosphopyridoxyl-D,L-7-azatryptophan, and suggest a new and easier route for synthesis and purification of 5'-phosphopyridoxyl-L-(or -D-)tryptophan. The absorbance and emission spectra of the 7-azatryptophan adduct are distinctly different than those of pyridoxal 5'-phosphate or the tryptophan adduct. We propose that 5'-phosphopyridoxyl-D,L-7-azatryptophan has great potential as an intrinsic probe in optical studies of protein dynamics.

Indicators and Reagents↗
PubMed1991

Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination.

Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.

Binding Sites↗
PubMed1988

Photophysics and reactivity of heme proteins: a femtosecond absorption study of hemoglobin, myoglobin, and protoheme.

On the basis of our time-resolved absorption measurements of hemoglobin (Hb), myoglobin (Mb), and protoheme (PTH), either unligated or ligated with CO, O2, or NO, we propose a description of the photophysics of heme proteins that encompasses their photodissociation, the origin and fate of the observed short-lived transients, and the appearance of the ground-state, unligated heme proteins. Two distinct species are formed upon ligand photodissociation, which occurs in less than 50 fs. We assign these species to excited states of the unligated heme and label them (for the case of hemoglobin) as Hb*I and Hb*II. We suggest that Hb*I is already at least partially domed and has a spin state of at least S = 1. Hb*I decays in 300 fs to the ground-state unligated heme species, which we consider to be S = 2 and at least partially domed. The population of Hb*II varies with the ligand. It is more significant when the ligand is O2 or NO than when the ligand is CO. The similarities of the picosecond and femtosecond bleaching and absorption kinetics of HbCO with those of PTHCO (and of HbNO with those of PTHNO) indicate that in this time domain the importance of steric features of the protein are less important than the nature of the ligand itself in the geminate recombination process as well as in the relative amounts of the two heme excited states created. It is suggested that the quantum yield of ligand photodissociation is unity whether the ligand is O2, NO, or CO. The low yield of photodissociated heme-O2 or heme-NO compounds as measured on the microsecond time scale is thus attributed to a fast (2.5 ps) recombination of O2 or NO with Hb*II. We discuss geminate recombination measurements of cyanomet hybrid hemoglobins with NO and consider these results in terms of alpha and beta subunit heterogeneity. The first picosecond transient absorption spectra of cyanomet-CO hybrid hemoglobins are presented and are compared with the spectra of other heme compounds. The superimposability of the transient spectra on the equilibrium spectra of heme compounds that exhibit minimal or no cooperativity is noted and is compared with the case of cooperative systems where the transient spectra are distorted with respect to the equilibrium spectra. This distortion is interpreted in terms of an interaction of a domed heme with the F helix.

Heme↗
PubMed1987

Time-resolved Raman spectroscopy with subpicosecond resolution: vibrational cooling and delocalization of strain energy in photodissociated (carbonmonoxy)hemoglobin.

A Raman spectrometer that provides both subpicosecond resolution and independent, tunable pump and probe pulses is described. The spectrometer is employed to obtain time-resolved spectra of (carbonmonoxy)hemoglobin (HbCO) at times from 0.2 to 95 ps subsequent to ligand photodissociation. The spectra are interpreted in terms of a vibrationally hot heme that cools substantially in 10 ps. Concomitant with the proposed vibrational cooling is a slower relaxation, which we suggest results from a protein response to heme doming induced by ligand detachment. Results and interpretations are discussed in the context of current models of the heme photophysics and of hemoglobin reactivity.

Adult↗