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Biomedical subjects

J Fidy

Publications and source records attributed to J Fidy.

31 records · Page 2Linked to original sources
PubMed1997

Interpretation of multiple Q(0,0) bands in the absorption spectrum of Mg-mesoporphyrin embedded in horseradish peroxidase.

Mg-mesoporphyrin horseradish peroxidase (MgMP-HRP) and MgMP-HRP complexed with naphtohydroxamic acid (NHA) have been studied by fluorescence line narrowing (FLN) and pressure tuning spectral hole burning (SHB) techniques. In each sample, the low temperature absorption spectra show more than one transition in the origin range of the Q band. Comparisons with broad-band fluorescence spectra and FLN studies suggest that the multiple band feature originates from the presence of different configurations of the metal-porphyrin that are subject to Qx-Qy splitting within the protein cavity. This suggestion is supported by pressure tuning SHB studies. In the uncomplexed as well as in the NHA-complexed form of MgMP-HRP, irradiation in the Q band produces photoproduct bands, which has been attributed to a species with smaller Qx-Qy splitting. In an amorphous matrix, on the other hand, only one form of MgMP could be found, and no splitting could be observed. The binding of NHA does not significantly alter the bulk parameters of the protein matrix, but it reduces the structural variety in the configuration of MgMP to a single form with a more distorted structure and thus with an enlarged Qx-Qy splitting.

Horseradish Peroxidase↗
PubMed1994

Electric field and conformational effects of cytochrome c and solvent on cytochrome c peroxidase studied by high-resolution fluorescence spectroscopy.

Electronic spectra of mesoporphyrin-substituted yeast cytochrome c peroxidase (MP-CcP) were measured as a function of pH, ionic strength, and binding of cytochrome c (cyt c) by fluorescence line narrowing (FLN) spectroscopy at 5 K. The FLN spectra provided information about the vibrational structure of the first excited singlet state of MP-CcP, the various tautomeric forms of mesoporphyrin, and the positions and widths of their 0,0 bands. The composite 0,0 band of MP-CcP at pH 6 could be resolved into three components with peak positions at 16,046, 16,103, and 16,203 cm-1. MP-CcP at pH 8 could be analyzed using two components with peak positions at 16,048 and 16,193 cm-1. The disappearance of the 16,103-cm-1 component at alkaline pH suggests that it is due to a "chemical substate" arising from protonation of His52 in the distal side of the porphyrin. Computer simulations of the electrostatic field that CcP imposes on its porphyrin show that, in the presence of charged axial histidines His52 and His175, the electrostatic field at porphyrin nitrogens increases, especially along the normal to the heme by about 200 mV/A. Electric field effects may account for pH-dependent spectral shifts of the 0,0 positions of the resolved components, although hydrogen bonding may also affect these positions. On the other hand, the peak position of the components was not affected by ionic strength or binding of cyt c, implying that the electrostatic field of the heme pocket of MP-CcP remains unchanged. Indeed, computed changes in ionic strength of the solvent show no modification of the electrostatic field at the porphyrin. The only detectable effect of ionic strength and binding of cyt c to MP-CcP is on the relative contributions of the components, suggesting some rearrangements in the vicinity of the heme. Finally, shifts in the position of the vibrational lines for MP-CcP components indicate either that the tautomers have different vibrational frequencies due to the nonsymmetry of the porphyrin and/or that tautomers experience various distortions. Comparison of the vibrational spectrum of the first excited singlet state of mesoporphyrin in CcP and horseradish peroxidase also suggests that the heme pocket in the two peroxidases provides different steric restrictions.

Binding Sites↗
PubMed1994

Spectral hole burning and selection of conformational substates in chromoproteins.

We investigated spectral holes burnt at 1.5 K into the origins of several tautomeric forms of mesoporphyrin IX-substituted horseradish peroxidase at pH 8 under pressures up to 2 MPa. From the pressure-induced lineshift the compressibility of the apoprotein could be determined. We found that the compressibility changed significantly when measured at different tautomer origins. It was concluded that there must be a correlation between the tautomer configurations of the chromophore and the actual structures of the apoprotein. As a consequence, specific conformational substates of the protein can be selected by optical selection of the associated tautomers.

Journal Article↗
PubMed1993

Protein conformation monitored by energy-selective optical spectroscopy.

Fluctuations in the polypeptide chain lead to disorder in proteins and to a distribution in the parameters that regulate their functions. Using low temperature (to reduce the fluctuations) and narrow-band lasers (to select one substate among the many forms), high-resolution absorption and fluorescence spectra for chromophores in proteins can be obtained. These spectra reveal information on the kind and extent of disorder in proteins and allow for the determination of the vibrational energies of both ground and excited state molecules, true inhomogeneous spectral width, and kinetic studies of individual protein substates.

Lasers↗
PubMed1992

More than two pyrrole tautomers of mesoporphyrin stabilized by a protein. High resolution optical spectroscopic study.

Mesoporphyrin IX substituted horseradish peroxidase was studied by fluorescence line narrowing and hole burning techniques at cryogenic temperatures. The spectral data reveal that four pyrrole tautomeric configurations of the chromophore are populated within the protein under the influence of irradiation and/or thermal treatment, and the existence of a fifth and a sixth tautomeric configuration is also likely. The relative population of the tautomers changes upon deuterium substitution through modification of the phototransition rate, and also depends on pH, which changes the protonation of neighboring amino acids in the heme pocket. The energy separation of the origins of the tautomers is approximately 100 cm-1. The distribution of barrier heights separating the different tautomeric forms in the ground state and their distribution was determined by temperature cycling hole burning. The distributions can be approximated by Gaussians. The experiment directly yields the distributions on a relative temperature scale, and a model is presented to transform the barrier heights into energy values. It is suggested that the energies for the tautomers are split partially due to the protein crystal field and that the trapping of the tautomeric forms is the consequence of interactions with neighboring amino acids within the heme pocket.

Biophysical Phenomena↗
PubMed1991

Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase.

For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features.

Horseradish Peroxidase↗
PubMed1989

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing.

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo-and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (alpha-, beta-, gamma-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0----S1 (0----0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by approximately 20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only beta-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0----0 transition energy for the tautomeric forms of MP and an increase in the 0----0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the delta v = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for beta-RE. In the same energy range, however, the transition into the delta nu = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA less than NHA less than HQ less than RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0----0 energies (from 60-70 to approximately 30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.

Energy Transfer↗
PubMed1986

Comparison of biophysical parameters of primary low and high metastatic Lewis lung tumor cells.

Lewis lung tumor cells from tumors of both high and low metastatic potential were isolated from the leg muscle of C57-B1 mice and were purified by density gradient centrifugation. The purified cells retained most of their tumor-forming capacity; however, the difference in the metastatic capacity of the 2 cell lines increased with the purification process. The purified and viable cells were subjected to different biophysical studies. Electron spin resonance studies showed that the motional freedom of lipid and protein molecules of the cellular membrane is higher in the low metastatic cell population. Both cell types reduce penetrating and non-penetrating free radicals, but the reducing ability of the low metastatic cell population is 4 times greater than that of the high metastatic cell line. These findings argue for increased membrane dynamics in the low metastatic cell population.

Animals↗
PubMed1983

Fluorescence label studies of the phase transitions of T7.

Optical density, fluorescence intensity, polarization and lifetime measurements were performed to analyze the temperature-induced phase transitions of phage T7 in several buffers. For labelling the intraphage DNA, ethidium bromide, proflavine and rivanol were applied for the proteins 1,6-diphenyl-1,3,5-hexatriene (DPH). In the temperature range of 20 degrees C-100 degrees C several structural changes of T7 were detected. Making corrections for light scattering by using integrating spheres and analyzing the fluorescence signals, the phase transition of intraphage DNA below 60 degrees C was interpreted as a superhelical relaxation phenomenon. The structural changes found by optical density at higher temperatures could be assigned to a change in the phage DNA or to a change in its protein part on the basis of fluorescence-melting results concerning DNA and protein labels. The effects of ionic strength and environment on the structural changes were studied.

Anti-Infective Agents, Local↗
PubMed1980

Chromatographic study of the UV photoproducts in uracil solid phase systems.

UV photoproducts were induced in two types of uracil solid phase systems: in evaporated and recrystallized thin layers and in dried aquaeous solution respectively. The nature of the photochemical reactions in the two systems was compared by gradient elution column chromatography. In the case of dried solution two types of cyclobutane dimers were isolated, but in recrystallized thin layers only one type was found. The efficiency of the reaction in evaporated layers was much higher than in those produced by drying. Based on the crystal structure of uracil and previous absorption spectroscopic studies of the photochemistry of thin crystalline layers it was stated, that the single type of photodimers produced in evaporated thin layers is characteristic for the photochemical reaction proceeding in crystal matrix; and its structure was proposed to be identical with that of the cis-syn stereoisomer dimer.

Photochemistry↗