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.

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.

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.

Effect of the central disulfide bond on the unfolding behavior of elongation factor Ts homodimer from Thermus thermophilus.

Functionally active elongation factor Ts (EF-Ts) from Thermus thermophilus forms a homodimer. The dimerization interface of EF-Ts is composed of two antiparallel beta-sheets that can be connected by an intermolecular disulfide bond. The stability of EF-Ts from T. thermophilus in the presence and absence of the intermolecular disulfide bond was studied by differential scanning calorimetry and circular dichroism. The ratio of the van't Hoff and calorimetric enthalpies, delta H(vH)/delta H(cal), indicates that EF-Ts undergoes thermal unfolding as a dimer independently of the presence or absence of the disulfide bond. This can be concluded from (1) the presence of residual secondary structure above the thermal transition temperature, (2) the absence of concentration dependence, which would be expected for dissociation of the dimer prior to unfolding of the monomers, and (3) a relatively low heat capacity change (delta Cp) upon unfolding. The retained dimeric structure of the thermally denatured state allowed for the determination of the effect of the intermolecular disulfide bond on the conformational stability of EF-Ts, which is deltadelta G(S-S,SH HS) = 10.5 kJ/mol per monomer at 72.5 degrees C. The possible physiological implications of the dimeric EF-Ts structure and of the intersubunit disulfide bond for the extreme conformational stability of proteins in thermophiles are discussed.

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.

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.

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.

Heat-induced conformational transition of cytochrome c observed by temperature gradient gel electrophoresis at acidic pH.

Temperature-gradient gel electrophoresis (TGGE) has been used to study the thermal unfolding of ferricytochrome c in low and high concentrations of acetic acid. It has been observed that the mobility of cytochrome c is a linear function of temperature when the system is characterized by a homogeneous population of conformation-state, single molecular species. Within the transition temperature range, the mobility clearly displays the characteristic sigmoidal shape describing the transitions of protein unfolding. The data obtained by TGGE were used to estimate the apparent thermodynamic parameters (enthalpy change deltaHvh and transition temperature Tm), associated with the transition of unfolding. The accuracy of the apparent thermodynamic parameters obtained by this method agrees within error limits with the values obtained by direct calorimetric measurements using differential scanning calorimetry (DSC).

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.

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 interfacial behavior of cytochrome c studied by pendant-drop technique.

The adsorption properties of cytochrome c (cyt c) were characterized by surface tension measurements using the pendant-drop method employing the digital image analysis of the drop shape. The method was applied to the study of the protein conformation change due to acidification at low ionic strength. The observation of the saturated steady-state surface tension shows that decrease in pH induces its cooperative change centered around pH 2.5. This value is equal to the value of apparent pK of the acid-induced transition of the horse ferricyt c from a native state to the unfolded conformation. This indicates that the saturated steady-state surface tension is sensitive to the conformation of cyt c in bulk phase, and the pendant-drop method might be used to monitor changes in the tertiary structure of proteins.

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.

Coulombic and noncoulombic effect of polyanions on cytochrome c structure.

The properties of the complexes of ferricytochrome c with two different polyanions--poly(vinylsulfate) and poly(4-styrene-sulfonate)--with a comparable charge density but with the different size of the uncharged part of their molecules have been studied by means of optical spectroscopy, differential scanning colorimetry, and gel chromatography. Ferriccytochrome c formed a complex with the former one through coulombic interactions and remained in a native-like state. The addition of the second polyanion to a solution of ferric cytochrome c at a low ionic strength, pH 7.0, resulted in profound conformational change in the hydrophobic core of protein (opening of the heme crevice with a perturbation of the methionine 80-heme iron bond and the hydrophobic core of the protein). These may be understood as an involvement of noncoulombic (hydrophobic, H-bonding) interactions of the uncharged part of the polyanion molecule. Conformational changes and the observed shift in acidic transition from low spin to high spin state of ferric cytochrome c detected in the presence of the polyanions may have biological implication in understanding the origin of conformational changes in proteins induced in the course of their interaction with membrane lipids and membrane proteins.

Dimers of Thermus thermophilus elongation factor Ts are required for its function as a nucleotide exchange factor of elongation factor Tu.

Elongation factor Ts (EF-Ts) promotes the formation of active GTP-bound elongation factor Tu (EF-Tu) by accelerating the dissociation of GDP from the EF-Tu x GDP complex. Thermus thermophilus EF-Ts forms a dimer in solution, which is stabilised by interaction of a three-stranded antiparallel beta-sheet from each of the two EF-Ts molecules. A disulfide bridge and several hydrophobic interactions are the main structural elements which stabilise the dimer [Jiang, Y., Nock, S., Nesper, M., Sprinzl, M. & Sigler, P. B. (1996) Biochemistry 35, 10269-10278]. Site-directed mutagenesis was used to study the dimer formation and the effect of dimerization on the nucleotide exchange activity. The presence of the covalent disulfide bridge between the Cys190 residues has no effect on the activity. However, this disulfide bridge is not a necessary condition for the activity of EF-Ts. The amino acid residues Leu73, Cys190 and Phe192 form a hydrophobic core on the dimerization interface. Their replacement by Asp, Ala and Asp, respectively, influences to different degrees the stability of EF-Ts, the ability of EF-Ts to form dimers, and the ability to interact with EF-Tu. EF-Ts variants which were unable to form dimers were also inactive in the nucleotide exchange on EF-Tu. CD spectroscopy confirmed that this loss of activity is not due to changes in EF-Ts secondary structure. By calorimetric measurements, it was demonstrated that the dimer formation considerably contributes to the thermostability of T. thermophilus EF-Ts. Dimerization of T. thermophilus EF-Ts is required to fulfil its physiological function in protein biosynthesis and probably represents a strategy of the translation system in this thermophile to withstand high temperatures. The biochemical data presented here are supported by the recently solved structure of the T. thermophilus EF-Tu x EF-Ts complex [Wang, Y., Jiang, Y., Meyering-Voss, M., Sprinzl, M. & Sigler, P. B. (1997) Nature Struct. Biol. 4, 650-656] in which each EF-Tu in the dyad symmetrical heterotetrameric complex interacts with two subunits of EF-Ts.

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 polyglutamate on the thermal stability of ferricytochrome c.

The effect of saturated solutions of polyglutamate on the thermal stabilities of the Met-80-heme iron bond and of the ferricytochrome c as a whole were studied by absorption spectroscopy and differential scanning calorimetry at pH 7.0. According to spectral data the midtransition temperature of the cleavage of the sulfur-iron bond was 57.4 +/- 0.5 degrees C and 66.8 +/- 0.5 degrees C for cytochrome c and cytochrome c-polyglutamate complex, respectively. Addition of polyglutamate to cytochrome c at pH 7.0 alters the denaturation properties of the protein. As follows from DSC scans, the denaturation temperature for cytochrome c is decreased from 85.4 +/- 0.2 degrees C to 68.7 +/- 0.2 degrees C in the presence of the saturated amount of polyglutamate. The protein stability in terms of Gibbs energy change at protein unfolding amount to delta G(25 degrees C) = 22.7 +/- 2.7 and 32.0 +/- 2.2 kJ/mol, for cytochrome c and cytochrome c-polyglutamate complex, respectively, at pH 7.0. It is evident that polyglutamate increases the thermal stability of the sulfur-iron bond and decreases the denaturation temperature of the cytochrome c molecule as a whole. The complex thermal stability in terms of Gibbs energy is not lower than that of cytochrome c in the range of physiological temperatures.

Interactions between heparinoids and alcohol dehydrogenase.

The interaction between polysulfated polysaecharides (low-molecular-weight heparin LMWH, dextran sulfate DS and pentosan sulfate PS) and yeast alcohol dehydrogenase (YADH) was investigated. The fluorescence and UV spectra of YADH after adding the tested polysaccharides have confirmed the interaction between the enzyme and these compounds. Kinetic studies have shown that LMWH, DS and PS are inhibitors of YADH (mixed type with respect to NAD). The most potent inhibitor is PS (ID50=37.5 ng/ml, Ki=0.6 muM). The inhibition effect depends on the ionic strength (the inhibition decreased by about 50% in the presence of 100 mM Na2SO4) and pH value (the inhibition decreased at pH>7). The results indicate that the inhibition effect of these polyanions is caused by their electrostatic interactions with the NAD-binding region of YADH.

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.