Reaction of superoxide anions with melanins: electron spin resonance and spin trapping studies

Scavenging of superoxide radicals by melanin is a possible factor in the photoprotection afforded by melanin pigments. The reaction between superoxide anions and melanins has been studied by electron spin resonance and spin trapping methods. It was found that superoxide anions react to produce melanin free radicals in a reaction inhibited by superoxide dismutase but not by catalase. The rate of radical formation depends on the concentration of melanin and superoxide, the pH of the medium and the presence of diamagnetic metal ions. The melanin pigment competes with the enzyme superoxide dismutase for removal of superoxide radicals. It was found that the xanthine-xanthine oxidase system is not suitable for studying the reaction of superoxide with melanin, as the enzymatic activity of xanthine oxidase is considerably inhibited by melanin.     

Radical anions from one-electron-reduced adrenochrome. Detection and identification by electron spin resonance spectroscopy

Free radicals from the one-electron reduction of adrenochrome have been studied in aqueous solutions. These radicals have been detected and identified by electron spin resonance spectroscopy, using spin stabilization methods (complexation with diamagnetic metal ions) to enhance radical concentrations. It is shown that the radicals have a characteristic ESR spectrum enabling their identification in complex systems. The spin density distribution in the radicals has been studied as a function of complexing metal ions and solvent composition. In the presence of oxidants (e.g., oxygen) the spectrum of the radical is replaced by that derived from the one-electron exidation of adrenochrome.     

Effect of proteolysis on the electron spin resonance spectra of maleimide spin labeled erythrocyte membrane

The ratio of low-field amplitudes of weakly and strongly immobilized signals of ESR spectra of a maleimide spin label bound to erythrocyte membranes (hw/hs) increases progressively during incubation at 37 degrees C. This increase is due to the 'self-digestion' of membrane proteins by endogenous proteinases and is attenuated by proteinase inhibitors. Digestion of membranes with chymotrypsin also increases the hw/hs ratio. These results suggest a need for a careful interpretation of data from spin-labeled membrane proteins, especially in experiments involving prolonged incubations of membrane preparations when the proteolytic effects may be significant.     

Electron spin relaxation of the electron paramagnetic resonance spectra of cytochrome c

The progressive power saturation of the electron paramagnetic resonance (EPR) spectrum of ferricytochrome c has been investigated in order to determine the spin-lattice relaxation time of the center. We have generalized the usual saturation treatments to include the effects of extended sample size and anisotropic g values as well as derivative spectra. We find that the results are consistent with a T7 power law in the temperature range 6--25 K. At temperatures above 25 K the relaxation time is too short for successful power saturation. Observation of the linewidth shows that the relaxation behavior continues as a first-order Raman process to 50 K.     

Reappraisal of the electron spin resonance spectra of maleimide and iodoacetamide spin labels in erythrocyte ghosts.

The effects of sulfhydryl inhibitors (iodoacetamide and N-ethylmaleimide) on the electron spin resonance spectra of two maleimide and two iodoacetamide spin labels in erythrocyte ghosts were found to correlate with their relative “lipid”/water partition coefficients. But the spectral characteristics of the maleimide spin labels, and their ghost concentrations after iodoacetamide inhibition, are not consistent with the hypothesis that interprets their spectra solely on the basis of a heterogenous membrane distribution. An alternative hypothesis is suggested which is compatible with relative “lipid solubilities” and the iodoacetamide inhibition spectra.     

Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O₂ half-saturation pressure, measured at 25 C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25 C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -log p_1/2 (O₂)/gDpH=0.36 mol H⁺/mol O₂ and amplitude of the Bohr-effect curve log p_1/2 (O₂)=0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O₂ affinities at 25 C which follows the order: proto < meso < deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with g=2.035 and by the low-field shift of g. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with g=2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O₂ affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in g. The correlation of the cobalt hyperfine constants of the oxy forms with the O₂ affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.     

Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O2 half-saturation pressure, measured at 25 degrees C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25 degrees C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -- deltalog p1/2 (O2)/deltapH = 0.36 mol H+/mol O2 and amplitude of the Bohr-effect curve deltalogp1/2 (O2) = 0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O2 affinities at 25 degrees C which follows the order: proto less than meso less than deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with gparallel = 2.035 and by the low-field shift of gparallel. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with gparallel - 2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O2 affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in gparallel. The correlation of the cobalt hyperfine constants of the oxy forms with the O2 affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.     

High-field electron spin resonance of spin labels in membranes

High-field electron spin resonance (ESR) spectroscopy is currently undergoing rapid development. This considerably increases the versatility of spin labelling which, at conventional field strengths, is already well established as a powerful physical technique in membrane biology. Among the unique advantages offered by high-field spectroscopy, particularly for spin-labelled lipids, are sensitivity to non-axial rotation and lateral ordering, a better orientational selection, an extended application to rotational dynamics, and an enhanced sensitivity to environmental polarity. These areas are treated in some depth, along with a detailed consideration of recent developments in the investigation of transmembrane polarity profiles.     

The electron spin resonance and absorption spectra of microsomal cytochrome P-450 and its isocyanide complexes

The absorption spectra of oxidized P-450-isocyanide complexes were the same in difference spectra irrespective of the isocyanide derivative tested. However, with these reduced P-450-isocyanide complexes, absorption at 455 mμ increased, and that at 430 mμ decreased, with increasing carbon atom number of the isocyanide derivative at a definite pH. The same changes were seen with individual complexes with increasing pH.

The dissociation constants of oxidized P-450-isocyanide complexes decreased with increase in carbon atom number of the isocyanide. These results were confirmed by electron spin resonance (ESR) spectroscopy. However, the dissociation constants of reduced P-450-isocyanide complexes were essentially identical and the dissociation constants of the oxidized and reduced P-450-isocyanide complexes were little affected by pH.

The oxidized P-450-isocyanide complexes gave magnetically specific ESR signals. The orbital energy differences of d orbitals of the heme iron of the complexes increased with increase in the carbon atom number of the isocyanide.

Purified P-450 and its isocyanide complexes were rapidly reduced by a ferredoxin-NADP⁺ reductase system.     

Effect of selected anions and solvents on the electron absorption, nuclear magnetic resonance, and infrared spectra of the N-retinylidene-n-butylammonium cation.

The specific conteranion and the solvent have been shown to regulate the electronic excitation energy of the N-retinylidene-n-butylammonium cation. Halogenated hydrocarbon solvents which can hydrogen bond with the anion shift the lambda-max to longer wavelengths, whereas the solvent dipole, acting as a bulk effect, shifts the wavelength-max to shorter wavelength. Here solvents which can donate two hydrogens for hydrogen bonding, such as cis- and trans-1,2-dichloroethylene and cis- and trans-1,2-dichlorocyclohexane, are used as solvents for the Cl-, Br-, and I- salts. As expected the cis solvents allow longer wavelengths than do the trans solvents. Results of nuclear magnetic resonance spectroscopy are shown to be in agreement with electronic absorption spectroscopy. The C-11 proton and the C-13 and C-9 methyl protons show a considerable downfield shift in the salts with respect to the Schiff base. Furthermore the same protons show a continuing downfield shift as the anions are exchanged from Cl-, Br- to I-. This is an agreement with the interpretation of greater positive charge delocalization as the anions are changed in the above manner. The infrared absorptions of the C - N group in the Schiff base and the protonated form are shown to be almost similar. This is rationalized by showing that the force constant can remain constant as the highly related factors bond order, bond distance, and the effective electronegativity are changed in a self-compensating manner.