Catalytic effects of silver plasmonic nanoparticles on the redox reaction leading to ABTS˙+ formation studied using UV-visible and Raman spectroscopy.

ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) is a compound extensively employed to evaluate the free radical trapping capacity of antioxidant agents and complex mixtures such as biological fluids or foods. This evaluation is usually performed by using a colourimetric experiment, where preformed ABTS radical cation (ABTS˙+) molecules are reduced in the presence of an antioxidant causing an intensity decrease of the specific ABTS˙+ UV-visible absorption bands. In this work we report a strong effect of silver plasmonic nanoparticles (Ag NPs) on ABTS leading to the formation of ABTS˙+. The reaction of ABTS with Ag NPs has been found to be dependent on the interfacial and plasmonic properties of NPs. Specifically, this reaction is pronounced in the presence of spherical nanoparticles prepared by the reduction of silver nitrate with hydroxylamine (AgH) and in the case of star-shaped silver nanoparticles (AgNS). On the other hand, spherical nanoparticles prepared by the reduction of silver nitrate with citrate apparently do not react with ABTS. Additionally, the formation of ABTS˙+ is investigated by surface-enhanced Raman scattering (SERS) and the assignment of the most intense vibrational bands of this compound is performed. The SERS technique enables us to detect this radical cation at very low concentrations of ABTS (∼2 µM). Altogether, these findings allow us to suggest the use of ABTS/Ag NPs-systems as reliable and easy going substrates to test the antioxidant capacity of various compounds, even at concentrations much lower than those usually used in the spectrophotometric assays. Moreover, we have suggested that ABTS could be employed as a suitable agent to investigate the interfacial and plasmonic properties of the metal nanoparticles and, thus, to characterize the nanoparticle metal systems employed for various purposes.

Interaction of cationic surfactants with DNA detected by spectroscopic and acoustic wave techniques.

Interaction of the cationic surfactants benzalkonium chloride and 1-hexadecylpyridinium chloride, in the concentration range 0.1 microM to 1 mM with calf thymus DNA and with short 19-mer double-stranded DNA has been examined in solution using UV absorption and fluorescent spectroscopies and at the liquid-solution interface by thickness-shear mode acoustic wave sensor. Higher concentrations of surfactant resulted in an increase of UV absorption, and decrease of melting temperature and van't Hoff enthalpy of calf thymus DNA. Both surfactants induce fluorescence quenching of ethidium bromide which is also associated with intercalation of the molecules into the nucleic acid strand. The effect of the pyridinium compound is greater than for the other surfactant likely because of the lower size of polar head group in this molecule. With respect to acoustic wave detection at the device surface, for relatively low surfactant concentrations (below 100 microM), decreases of both series resonant frequency and motional resistance were observed. At higher surfactant concentration both parameters increased. These effects are attributed to acoustic coupling processes that occur at the device-film/liquid boundary.

Influence of NaCl and sorbitol on the stability of conformations of cytochrome c.

Influence of ionic (NaCl) and non-ionic (sorbitol) additives on structural transitions of cytochrome c was investigated by circular dichroism, optical and EPR spectroscopy. Transformations of cytochrome c, induced by the acidification of solution and temperature perturbation, were monitored in the heme pocket together with changes in the secondary structure. NaCl and sorbitol exhibited antagonistic effect on the acid-induced transition of the protein. Sorbitol enhanced the stability of native conformation while NaCl destabilized this state. The midpoints of acid-induced transitions in the axial coordination of heme as well as in the secondary structure occurred nearly at the same pH values. However, temperature-induced transitions in the unfolding of the secondary structure were almost coincidental with the cleavage of Met80-Fe bond only in the sorbitol solutions. In the salt solution the Met80-Fe bond was markedly more labile than the secondary structure.

Early melting of supercoiled DNA topoisomers observed by TGGE.

We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity (sigma). We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs. The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity. Supercoiled topo-isomers represent a system of molecules that is sensitive to changes in temperature. We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values. Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress. The estimated melting temperature for plasmids with sigma < -0.05 is very significant because these temperatures for early melting are below physiological temperatures.

Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH.

The effect of polyanion, poly(vinylsulfate), used as a model of negatively charged surface, on ferric cytochrome c (ferricyt c) structure in acidic pH has been studied by absorbance spectroscopy, circular dichroism (CD), tryptophan (Trp) fluorescence and microcalorimetry. The polyanion induced only small changes in the native structure of the protein at neutral pH, but it profoundly shifted the acid induced high spin state of the heme in the active center of cyt c to a more neutral pH region. Cooperativity of the acidic transition of ferricyt c in the presence of the polyanion was disturbed, in comparison with uncomplexed protein, as followed from different apparent pK(a) values observed in a distinct regions of the ferricyt c electronic absorbance spectrum (4.55+/-0.08 in the 620 nm band region and 5.47+/-0.15 in the Soret region). The ferricyt c structure in the complex with the polyanion at acidic pH (below pH 5.0) has properties of a molten globule-like state. Its tertiary structure is strongly disturbed according to CD and microcalorimetry measurements; however, its secondary structure, from CD, is still native-like and ferricyt c is in a compact state as evidenced by quenched Trp fluorescence. These findings are discussed in the context of the molten globule state of proteins induced on a negatively charged membrane surface under physiological conditions.

Spectrophotometric detection of the interaction between cytochrome c and heparin.

Heparin inhibits transport of electrons from reduced cytochrome c to cytochrome c oxidase. The effect is due to the interaction of heparin with cytochrome c. It has been observed that binding of heparin to the reduced or oxidized cytochrome c changes the spectrum of cytochrome c at the Soret region. Affinity chromatography of heparin in cytochrome c immobilized to thiol-Sepharose shows that commercial heparin is eluted in the low-affinity and high-affinity fractions. Both participate in the interaction with cytochrome c. Polylysine induces decay of the cytochrome c-heparin complex.

Interaction of alkaline phosphatase with cytochrome c.

Alkaline phosphatase (AP) a protein which exhibits long-lived phosphorescence lifetime and ferricytochrome c as a phosphorescence quenching agent were examined. The excitation of the tryptophan triplet state resulted in cytochrome c reduction confirming long-range electron transfer as the quenching mechanism. The rate of electron transfer was not related to the length of the illumination interval; an additional reaction between the two proteins leading to cytochrome c reduction was detected. The reaction which proceeded in the dark was not sensitive to oxygen, was dependent on pH, and on the AP to cytochrome c ratio. At optimum 68 +/- 4% of the total cytochrome c could be reduced due to the presence of AP. On incubation of the two proteins the conformation of cytochrome c was altered as was evidenced by its decreased reducibility by ascorbate, by the disappearance of the absorption band at 695 nm, by the appearance of the new band at 620-640 nm, and by a change in circular dichroism spectra witnessing a structural alteration in the vicinity of the heme cleft. This was characterized by a profound increase in positive elipticity at 400 nm and by a reversible change in the magnitude of negative elipticity at 417 nm. The reaction was not significantly affected by the addition of sulfhydryl-binding and metal-complexing agents.

Quantitative structure-activity relationship of carbonylcyanide phenylhydrazones as uncouplers of mitochondrial oxidative phosphorylation.

The dependence of the uncoupling activity in the series of 16 carbonylcyanide phenylhydrazones on their physico-chemical properties (partition coefficient, dissociation constant and rate constant for reaction with thiols) is investigated using two physiologically based models, one for protonophoric mechanism of uncoupling and the other assuming the covalent modification of a membrane constituent to be the key step in this process. As indicated by uptake experiments, at the given conditions a lipophilic-hydrophilic equilibrium is attained without any loss of the compounds via chemical reactions. Using this fact to reduce the number of adjustable parameters, a better fit to the data on stimulation of respiration is obtained with the former (protonophoric) model.

Interaction of ferricytochrome c with polyanion Nafion.

The properties of the complex of ferricyt c with fluorosulfonated polyanion Nafion (as a representative 'hydrophobic' polyanion) have been studied by means of optical spectroscopy and differential scanning calorimetry. The addition of the polyanion to a solution of ferricyt c (pH 7.4) resulted in an expansion of the protein molecule characterized by a profound decrease in enthalpy of the thermal transition of ferricyt c. The conformational change of ferricyt c upon addition of Nafion was shown by a perturbation of the Met-80-heme iron bond and an apparent increase in the distance of Trp-59 from the heme. The conformational change in the heme region was also observed by examining the CD spectra. The conformational state of ferricyt c in a complex with Nafion was similar to that designated as state II by Hildebrandt (Hildebrandt, P. (1990) Biochim. Biophys. Acta 1040, 175-186) in the complex with negatively charged heteropolytungstates-a six-coordinated low-spin state with a destabilized structure of the heme crevice. The decrease in enthalpy of the thermal transition of ferricyt c, the spectral changes in absorbance and the CD spectra, together with an increase in Trp fluorescence induced by Nafion addition observed at high ionic strength, all point to the involvement of the non-coulombic interaction.

Effect of N-domain on the stability of elongation factor Ts from Thermus thermophilus.

Elongation factor Ts (EF-Ts) from Thermus thermophilus forms a stable, functionally active homodimer in solution. Its monomer is composed of two domains: amino-terminal domain containing 50 amino acid residues and a larger, 146 residues long, C-domain which participates in dimerization of EF-Ts. Effect of removal of the N-domain on the conformational stability of EF-Ts has been studied. For comparison, the stabilities of both the full-length EF-Ts and its C-domain were studied by differential scanning calorimetry, electronic absorption and fluorescence spectroscopies over a pH range from 4 to approximately 13. Thermal denaturation of EF-Ts and of C-domain, followed by circular dichroism at 222 nm, at pH 7.0, and the pH dependence of the fluorescence of the single tryptophan 30 residue indicate a conformational instability of the N-domain. While N-domain does not affect the stability of full-length EF-Ts at acidic pH, its removal leads to stabilization of the rest of the protein at basic pH. This is reflected by higher values of transition temperatures and calorimetric enthalpies of C-domain as compared to the full-length EF-Ts. High mobility of the N-domain in alkaline pH conditions decreased the thermal stability of covalently linked C-domain of EF-Ts. An increase in intramolecular interactions at acidic pH together with a decrease of conformational entropies of the thermally denatured proteins most likely diminishes this destabilization effect.

Effect of ionic strength on the interfacial properties of cytochrome c.

The surface tension behaviour of oxidised cytochrome c (cyt c) solution was characterised at various pH and ionic strength at the air/water interface. The pendant drop method employing digital image analysis of the drop shape was applied to the measurement of the surface tension. The adsorption properties of cyt c were utilised to study the protein conformation change effected by acidification and ionic strength. At high ionic strength, the saturated steady-state surface tension shows a cooperative change centred around 3.6 induced by a decrease in pH. Using spectroscopic experiments, the apparent pK of the acid-induced transition of horse cyt c from the native to the molten globular state is equal to 3.5. This fact indicates that the saturated steady-state surface tension is a parameter which might be used to monitor conformation changes of cyt c.

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry.

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.

Interaction of cupric ion with parvalbumin.

Cod parvalbumin, a calcium-binding protein, possesses a specific Zn2+ (or Cu2+) binding site per molecule. This work employed fluorescence energy transfer techniques to measure the distance between the Zn2+ (Cu2+) site and the stronger Ca(2+)-binding site in parvalbumin. Specifically, the distance between Tb3+ bound at the Ca2+ site and Co2+ bound to the Zn2+ (Cu2+) binding site was 10.3 +/- 0.9 A. Lastly, the effects of Cu2+ on the physico-chemical properties of parvalbumin were studied by measuring the accessibility of protein thiol groups to 5,5'-dithio bis(2-nitrobenzoic acid) and by its affinity for the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulfonic acid] dipotassium salt. The thiol group accessibility decreased and the affinity to the fluorescent probe increased upon complexation of Cu2+ to the protein. It appears that the binding of Cu2+ converts parvalbumin to an apo-like state.

Effect of polyanion on the acidic conformational transition of native and denatured ferricytochrome c. Circular dichroism study.

Interaction of polyanion poly(vinylsulfate) with oxidized cytochrome c (cyt c) significantly affects the protein main characteristics. One of them, pKa value of acidic transition, was shifted from an apparent pKa value 2.5 (typical for cyt c in low ionic strength solvent) to approximately 5.20 +/- 0.15 upon polyanion binding to the protein, pointing to a likely involvement of histidines 26 and/or 33 in the protein acidic transition in complex with the polyanion. The acidic transition followed at 6 different wavelengths all over circular dichroism spectrum, monitoring different parts of the protein structure, revealed basically two-state character process. Only ellipticity at 262 nm indicated a low-cooperative pH-induced conformational transition in heme region with an apparent pKa approximately 4.34 +/- 0.25 in accordance with absorbance change at 620 nm. Polyanion also interacts with chemically-denatured (in the presence of 9 mol/l urea) state of the protein as it follows from stabilization of protein residual structure at acidic pH and its effect on pKa value of acidic transition of chemically-denatured cyt c. Destabilization effect of polyanions on native and, on the other hand, stabilization influence on partially unfolded conformations of the protein are discussed with an implication for their chaperone-like properties in vivo and in vitro.

Studies on interactions between metmyoglobin and heparin.

The complex formation between metmyoglobin and heparin was investigated by absorbance and fluorescence spectroscopy as well as differential scanning microcalorimetry. In acidic pH region, three distinct complexes detected by absorbance measurements are formed depending on pH and time of equilibration. The kinetics of the conformational transition of metmyoglobin-heparin complex equilibrated at neutral pH observed after pH change to acidic region comprises two steps. During the first step, characterized by rapid changes of the absorption spectra (approximately 5 minutes) as well as fluorescence intensities, reversible transition with pK = 6.5 +/- 0.1 occurs and the first type of the complex forms. Below pH 6.2 the transition with pK = 5.7 +/- 0.1 is observed and the second type of the complex is formed. During the second slow step, the third type of the complex formed after 30 minutes of equilibration is characterized by a spectrum corresponding to low-spin form without protein axial ligand bound. At neutral pH and 25 degrees C, the interaction between metMb and heparin only slightly alters absorption and fluorescence spectra. On the other hand, the formation of metMb-heparin complex is established from the decrease of the transition temperature from 80.4 +/- 0.5 degrees C to 74.7 +/- 0.5 degrees C. Moreover, the binding of heparin prevents the aggregation of the protein at isoelectric point resulting in a considerable increase in the reversibility of thermal denaturation.

Effects of N-tricyanovinylamines on oxidative phosphorylation and level of SH-groups in rat liver mitochondria.

The effects of N-substituted tricyanovinylamines on oxidative phosphorylation as well as on glutathione and total SH group concentrations in rat liver mitochondria was studied. The N-TCVA derivatives studied (N-cyclohexyl; N-isobutyl; N-benzyl; N-phenyl; N-4-Br-phenyl; N-3-nitrophenyl) had an uncoupling effection on the oxidative phosphorylation. They stimulated the respiration of mitochondria and influenced their membrane potential. In their property as SH agents, the N-TCVA derivatives reduced the level of TSH groups of the mitochondria present in concentrations of 2 mumol/mg protein. The activity of succinate dehydrogenase was decreased by N-TCVA by 13%. N-TCVA derivatives changed the redox state of glutathione in mitochondria. This effect was observed at the concentration 0.3 mumol/mg protein. The results obtained in the present study support the view that the glutathione status is more sensitive than the total level of SH groups to incubation of mitochondria with SH agents such as N-TCVA derivatives.

Effect of nucleotides on thermal stability of ferricytochrome C.

The effect of nucleotides on the structure and thermal stability of ferricytochrome c was studied by differential scanning calorimetry. The association of cytochrome c with ATP and ADP resulted in a decrease in the denaturation temperature of cytochrome c by 7 degrees C and 4 degrees C, respectively, at pH 7.0. AMP did not change the denaturation temperature of cytochrome c at pH 7.0. The ratio between van't Hoff and calorimetric enthalpy of denaturation accounts for the fact that cooperative denaturation of 3-4 molecules of cytochrome c occurred in the presence of ATP at the pH range from 5 to 9. ADP gave rise to the interaction of 2-3 molecules of ferricytochrome c at pH 6-7.5, and AMP did not affect the interaction of protein molecules. Cytochrome c alone also associated at pH 7.5-10. At physiological ionic strength, pH 7.0, only ATP induced an association of ferricytochrome c molecules. No intermolecular interaction of ferricytochrome c molecules was observed at concentrations of NaCl higher than 0.2 mol/l not even in the presence of ATP.

Study of the complex formation between amine local anesthetics and uncouplers of oxidative phosphorylation carbonyl cyanide phenylhydrazones.

Spectroscopic evidence is presented which indicates that the anionic uncoupler carbonyl cyanide-4-nitro-2-chloro-phenylhydrazone and the amine local anesthetics form a complex in aqueous solution. The complex formation studies were carried out for several pharmacologically important tertiary amines and some primary amines. Their relative potencies to form a complex with uncoupler have followed the order: procaine < trimecaine < tetracaine < dibucaine < dodecylamine < dicyclohexylamine < hexadecylamine. As to the more lipophilic nature of the complex the emphasized penetration into octanol and reinforced retention into mitochondria was observed. The higher ability of the complex to colapse the mitochondrial membrane potential confirms this fact. The effective concentration of amine local anesthetics to form a complex was correlated with their physicochemical properties namely lipophilicity and acidobasicity. The highest effectivities for complex formation is shown by the most lipophilic and the most ionized molecules of amines. Present results point to the importance of considering the role of amine anesthetic-uncoupler complex in interpreting physiological or ion transport data in which these substances have been used together.

Malate dehydrogenases--structure and function.

Malate dehydrogenases (MDH, L-malate:NAD oxidoreductase, EC 1.1.1.37), catalyze the NAD/NADH-dependent interconversion of the substrates malate and oxaloacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane, and in the tricarboxylic acid cycle within the mitochondrial matrix. They are homodimeric molecules in most organisms, including all eukaryots and the most bacterial species. The enzymes share a common catalytic mechanism and their kinetic properties are similar, which demonstrates a high degree of structural similarity. The three-dimensional structures and elements essential for catalysis are conserved between mitochondrial and cytoplasmic forms of MDH in eukaryotic cells even though these isoenzymes are only marginally related at the level of primary structure.

Absorption and Raman spectroscopy study of cyt c-thiol complexes in acidic solutions.

Absorption UV-VIS and pre-resonance Raman spectra of acidic cyt c solutions with a series of thiols added (thiophenol, n-propanethiol, isopropanethiol, L-cysteine, dithiothreitol, 2-mercaptoethanol, N-acetyl-L-cysteine, p-acetamidothiophenol, 2-mercaptoethanamine, thioglycolic acid and mercaptopropionic acid), are presented. Interactions of cyt c molecule with the thiols were studied with the aim to identify binding of the thiols with the cyt c heme as its iron axial ligands. Absorption and Raman spectra showed some correlation between maxima of 700 nm region absorption band (typical for Fe-S axial bond in cyt c heme) and also wave numbers of spin state marker and axial ligand sensitive Raman bands on one, and pKa constant values of appropriate thiols on the other hand. These results imply thiol replacement of Met-80 from axial bond with heme iron and suggest that the force of Fe-L-cysteine axial bond is very close to the native axial bond (Fe-Met) for cyt c in neutral solution.