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K G Paul

Publications and source records attributed to K G Paul.

At least 19 recordsLinked to original sources
PubMed1991

Fast heme release from inactive proteins.

Kinetics for the release of the prosthetic group from hemoproteins is presented. Heme-protein separation is a biphasic reaction, where an initial phase is significantly faster than the dominant, slow phase. A previous communication concluded that the slow phase represents the active protein. This communication presents data for porphyrin release which shows that the initial fast phase represents an inactive form of the protein. Moreover, we suggest that the fast to slow phase ratio is a sensitive monitor of sample quality for many hemoproteins and that an extrapolation of the slow phase absorbance leads to new estimates for the true physical parameters of unperturbed proteins.

Chloride Peroxidase↗
PubMed1991

Heme-protein fission under nondenaturing conditions.

Slow heme transfer from horseradish peroxidases C2 and A2, cytochrome c peroxidase, chloroperoxidase, and leghemoglobins to a heme acceptor protein, apomyoglobin, has been studied under mild conditions. The reaction is best described as heme release into water followed by quick engulfment by apomyoglobin. The energetics of the activated process are large and interpreted as connected to both polypeptide motions during release and the ordering of water around the heme during solvation. The free energy required to break the iron(III)-ligand 5 (L5) bond is a minor but crucial portion of the activation free energy. Donor-acceptor protein interactions are not involved in the transfer. Fast heme release from inactive protein has also been observed. Apoprotein recombination with porphyrins and hemes suggest that this lack of activity is a result of Fe-L5 bond breaking.

Apoproteins↗
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↗
PubMed1987

Peroxidases: past and present.

The history of peroxidases spans nearly two centuries. Our knowledge has developed from early phenomenological observations of the colored products of peroxidase-catalyzed reactions, to our present understanding of many of the steps in the complex peroxidation reaction mechanism. Peroxidases are ubiquitous in plant and animal tissues and occur in diverse structural forms. Collectively, they are able to catalyze the hydroperoxide oxidation of many different kinds of organic and inorganic compounds. In spite of the great diversity of structures and functions, mechanisms of heme-containing peroxidases have several common features: (i) the transfer of the oxidizing equivalents of the hydroperoxidase to the enzyme to form Compound I, (ii) the reduction of Compound I by the transfer of electrons from donor molecules, (iii) the inactivation of Compound I by excess hydroperoxide. Rate constants for these and other steps in the peroxidation mechanism, as well as redox potentials, have been reported for many peroxidases. The molecular basis for the donor specificity of peroxidases has not yet been elucidated. Today, much interest is directed towards the biological functions of peroxidases and their reaction products.

Chemistry↗
PubMed1986

A 13C-NMR study of mutant hemoglobins with altered oxygen affinity.

The 13CO-NMR spectra of carbonylhemoglobins Saint Mandé (beta 102Asn----Tyr), Malmö (beta 97His----Gln), Hôtel Dieu (beta 99Asp----Gly) and Ao have been determined. The positions of the 13CO resonances for hemoglobins Ao, Malmö and Hôtel Dieu were similar indicating similar ligand environments for all. The 13CO resonance for the beta-subunit of Saint Mandé was upfield-shifted compared to the others. This is evidence that structural changes at the beta 102 position directly affect iron-ligand bonding as well as quaternary structure.

Carbon Isotopes↗
PubMed1986

Tryptophan in horseradish peroxidase.

Fluorescent derivatives of horseradish peroxidase C were prepared by replacing protoheme by protoporphyrin or mesoporphyrin. Calculations according to Förster on energy transfer allowed the determination of the distances of greater than 2.2 nm between tryptophan and porphyrin (heme) and greater than 2 nm between tryptophan and substrate-binding site. The modification of the single tryptophan with 2-hydroxy-5-nitrobenzyl bromide (Koshland's reagent) did not affect the enzyme's activity towards hydrogen peroxide or ascorbate. Modified and unmodified peroxidase showed the same affinity for aromatic substrates.

Energy Transfer↗
PubMed1985

Site-selected fluorescence spectra of porphyrin derivatives of heme proteins.

The emission spectra of the porphyrin in metal-free and Zn cytochrome c and in metal-free mesoporphyrin derivatives of horseradish peroxidases A and C, leghemoglobin, and myoglobin were examined as a function of temperature and excitation wavelength. At room temperature, the emission spectra were unresolved and were independent of excitation wavelength. At low temperature (4.2 K), the spectra depended upon excitation wavelength: using narrow-band excitation into the high-energy side of the 0-1 and 0-0 bands gave unresolved emission spectra whereas excitation into the low-energy side produced quasi-line spectra. The resolved spectra were different for the five proteins and further varied with pH, indicating chromophore-protein interactions. The spectra are interpreted in terms of site selection and phonon interactions.

Cytochrome c Group↗
PubMed1985

The vibrational bands of carbon monoxide bound to hemes or metal surfaces.

Infrared spectra of imidazole carbonyl complexes of 2,4-substituted hemes are presented. An increased CO stretch frequency is accompanied by a lowered FeC vibrational energy. Hartree-Fock-Slater electron structure calculations discern pi and sigma contributions to the observed shifts of vibrational energies. We conclude that an enhanced electron availability manifests itself as a lowered ferric/ferrous reduction potential, increased filling of the 2 pi orbital of liganded CO which in turn reduces nu CO and increases nu Fec, and increased basicity of the liganded CO. Analogies between CO liganded to heme and CO adsorbed onto metal surfaces are discussed.

Carbon Monoxide↗
PubMed1985

Spin and electron distributions in heme-cyanide models and hemeproteins.

Proton NMR spectra of low-spin Fe(III) cyanoprotoheme as prosthetic group in a number of proteins are presented. The diagonally positioned 1-, 5- and 3-, 8-methyl groups obey shifts proportional to the Fe(III)/(II) reduction potential Em7, which indicates a pseudo-contact interaction. The correlation with Em7 is understandable if one postulates an enhanced rhombic distortion, dominating the Fe-methyl dipolar interactions. Hartree-Fock-Slater quantum chemical calculations show no significant changes of spin density as a function of the Fe-L5 distance, except at the iron atom and predominantly in the 3dxz and 3dyz orbitals. 4p orbitals, on the other hand, uphold most of the changes of electron density. We also observe a principal difference in the amino acid sequences in the heme-accommodating pocket of oxygen carriers and two-electron transmitters.

Cyanides↗
PubMed1985

The transformation of chlorophenols by lactoperoxidase.

The lactoperoxidase-catalyzed transformations of penta-,2,3,4,6-tetra-, 2,4,6-tri-, 2,4-di- and 4-monochlorophenol were followed spectrophotometrically. Apparent stoichiometries of chlorophenol:H2O2 ranged from 1:1 for the tri- and tetrachlorophenol at pH7 to 5:2 for pentachlorophenol at pH 4. The initial velocity (v0) was only slightly influenced by changes in [H2O2] greater then 5 microns. v0 responded to [chlorophenol] according to the empirical expression v0=[lactoperoxidase] . (k1[chlorophenol] + k2[chlorophenol]2). The constant k1 was trichlorophenol, respectively, at pH 7. With the di- and monochlorophenol the solution soon became opaque, and the reaction ceased. The results show that more than one reaction occurs. Some comparisons were also made with horseradish peroxidase A and C. Cetyltrimethylammonium bromide prevented opaqueness, but was shown to be a substrate for lactoperoxidase. Assuming an average concentration of 0.1 microns for H2O2 and pentachlorophenol in man, the metabolic rate becomes 30 ng/h per g of peroxidase-containing tissue, possibly with deposition of the products.

Animals↗
PubMed1984

Mutant hemoglobin stability depends upon location and nature of single point mutation.

The temperature dependence of the rates of heme release from the beta subunits of methemoglobin A and 5 beta mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the alpha 1/beta 2 interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.

Heme↗
PubMed1984

X-ray absorption studies of intermediates in peroxidase activity.

The structures of the enzyme-substrate compounds of peroxidases and catalase determined by X-ray absorption spectroscopy are presented. The valence state of the iron in Compounds I and II is determined from the edge to be higher than Fe+3. A short Fe-Ne (proximal histidine) distance is observed in all forms except Compound II, forcing the Fe-Np average distance to be long, a result which differentiates the peroxidases from the oxygen transport hemoproteins and plays a pivotal role in the mechanism. A correlation is shown between the ratio of peaks in the low k (ligand field indicator ratio) region, the Fe-Np (heme pyrrole nitrogen) average distance, and the magnetic susceptibility, which provides a sensitive indicator of spin state. The mechanism of H2O2 reduction is shown by analysis of the structural changes observed in the intermediates. Possible relationship of these compounds to that of the peroxidatic form of cytochrome oxidase is suggested by these results.

Catalase↗
PubMed1983

Peroxidase activity and thiocyanate accumulation in salivary glands.

Salivary glands with high, low, or no peroxidase activity do not differ in [S14CN-] after the i.v. injection of KS14CN, nor do the glands differ from blood and muscle in [S14CN-]. The content of SCN- in a salivary gland does not mirror the gland's participation in the peroxidase-mediated antimicrobial mechanism.

Animals↗
PubMed1983

Ab initio calculations of electron distributions in heme-CO models.

We have, by the use of ab initio calculations, found a back-bonding state of pi symmetry close to the Fermi level for CO bound to FeN5C14. We thus find it likely that small shifts of the redox potential magnitude of EF - EV magnitude of will cause relatively large changes of the CO vibrational frequency. The separation of Fe 3d orbitals in our heme model is found to agree with what is predicted by ligand field theory for Oh symmetry. This paper presents nonrelativistic Hartree-Fock-Slater calculations of the 5 sigma bonding and 2 pi back-bonding between CO and Fe. The effects of up to 19 additional atoms are discussed for models of heme (COFe to COFeN5C14). The filled back-bonding state is found to be strongly influenced by second nearest neighbor atoms. By use of symmetry orbitals we have resolved the Fe 3d orbitals into the T2g and Eg representations of the Oh point group and find the former states to be occupied whereas the latter are unoccupied. The difference in occupancy is reduced when the CO ligand is removed which also causes an increased density of states at the Fermi level, i.e., the highest occupied and lowest unoccupied orbitals. Possible correlations between our data and experimental results are discussed for heme proteins as well as for metal surfaces.

Carbon Monoxide↗
PubMed1983

The reduction potential of lactoperoxidase.

The reduction potential of Fe(III)/Fe(II) lactoperoxidase has been determined. Optical determinations of equilibria with 2-methyl-3-hydroxy-1,4-naphthoquinone as indicator and prereduced 9,10-anthraquinone-2-sulfonate as reducing agent gave Em,7.0 = -191 +/- 2 mV. Potentiometric determinations with 9,10-anthraquinone-2-sulfonate as mediator and, in the reduced form as reducing agent, gave Em,7.0 = -188 +/- 1 mV. Addition of 0.5% N-cetyl-N,N,N-trimethyl-ammonium bromide, and using dithionite as reducing agent, gave Em,7.0 = -183 mV and -179 mV with 9,10-anthraquinone-2-sulfonate and 9,10-anthraquinone-2,6-disulfonate, respectively, as mediators.

Animals↗
PubMed1983

The mechanism of spontaneous heme release from horseradish peroxidase isoenzyme A2.

We have investigated the spontaneous release of heme from chromatographically homogeneous horseradish peroxidase A2 at varying temperature, pH and iron ligands. A "biphasic" rate of heme release was observed under all conditions. Upon further purification of HRP A2, a component was isolated that was homogeneous by polyacrylamide gel electrophoresis and exhibited a single first order rate of heme release. The rate of the release increased with pH and temperature, decreased in the presence of cyanide and increased slightly in the presence of fluoride. These results are consistent with the idea that the rate of heme release is a measure of the flexibility of the protein lining the heme "pocket" with iron bonding playing a secondary, though important role in heme-protein interactions.

Animals↗