pH-dependent conformational changes of ferricytochrome c induced by electrode surface microstructure

pH-dependent processes of bovine heart ferricytochrome c have been investigated by electronic absorption and circular dichroism (CD) spectra at functionalized single-wall carbon 'nanotubes (SWNTs) modified glass carbon electrode (SWNTs/ GCE) using a long optical path thin layer cell. These methods enabled the pH-dependent conformational changes arising from the heme structure change to be monitored. The spectra obtained at functionalized SWNTs/GCE reflect electrode surface microstructure-dependent changes for pH-induced protein conformation, pK(a) of alkaline transition and structural microenvironment of the ferricytochrome c heme. pH-dependent conformational distribution curves of ferricytochrome c obtained by analysis of in situ CD spectra using singular value decomposition least square (SVDLS) method show that the functionalized SWNTs can retain native conformational stability of ferricytochrome c during alkaline transition.

STABILITY AND FOLDING OF MUTANT FORMS OF ISO-1 CYTOCHROME-C FROM SACCHAROMYCES CEREVISIAE

Partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae were obtained by replacements of the evolutionarily conserved proline 71 with valine, isoleucine and threonine (Ernst et.al.,1985). Pro-71 lies at the juncture of two short helical regions and is believed to be important for proper local polypeptide chain folding within the iso-1-cytochrome c structure.^ To study folding in the absence of intermolecular disulfide dimer formation the free sulfhydryl group of Cys-102 was modified in both wild type and mutant proteins with an alkylating reagent, methyl methanethiosulfonate. Spectral analysis of the wild type and mutant proteins shows that the native-like functional (or partially functional) folded structure of cytochrome c is retained in the chemically modified derivatives. The replacement of Pro-71 with valine, isoleucine or threonine reduces the intensity of the 696 nm absorbance band which is an indicator of the Met-80 ligation to the heme. Thermal stability and guanidine hydrochloride unfolding studies of the mutant proteins shows a destabilization of the protein as a result of mutation. The degree of destabilization depends on the chemical nature of the substituent amino acid in the mutant protiens.^ Kinetics of folding/unfolding reactions of the proteins were monitored by fluorescence changes using stopped flow mixing to obtain guanidine hydrochloride concentration jumps ending below, within, and above the transition zone. The replacement of Pro-71 alters the rate on one of the fastest phases, $\tau\sb3$, while the two other phases, $\tau\sb1$ & $\tau\sb2$, remain the same.^ Slow refolding kinetic studies indicate that replacement of Pro-71 does not completely eliminate the absorbance or fluorescence detected slow phases leading to the conclusion that Pro-71 is not involved in the generation of the slow phases in the folding kinetics of iso-1-cytochrome c.^ The alkaline conformational change involving the disappearance of the 696 nm absorbance band occurs with increasing pH in the alkaline pH region (Davis et al., 1974). The apparent pK of this conformational change in mutant proteins is shifted as much as two pH units compared to wild type. The equilibrium and kinetic data of alkaline transition for the wild type follows a simple mechanism proposed by Davis et al., (1974) for horse heart cytochrome c. A more complex mechanism is proposed for the behavior of the mutant proteins. ^

Endonuclease Active Site Plasticity Allows DNA Cleavage with Diverse Alkaline Earth and Transition Metal Ions

A majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion.

The electrical and magnetic properties of lanthanide alkaline-earth transition-metal oxides

Five Ln(2)SrMCuO(6.5) oxides (M = Co, Ln = Y and Ho; M = Fe, Ln = Y, Ho, and Dy) were synthesized, and their crystal structures, IR spectra, and physical properties were studied. They have almost the same structure and crystallize in orthorhombic systems. Below room temperature, Y2SrFeCuO6.5, a known layered oxide, shows antiferromagnetic behavior, but the four new oxides are paramagnetic. Y2SrFeCuO6.5 fits the Curie-Weiss law in the temperature range 300-100 K, but Y2SrCoCuO6.5 shows complex magnetic behavior because of the disproportion of some Co+3 to Co+2 and Co+4 The five oxides are all p-type semiconductors in the measured temperature range and have large electrical resistivities at room temperature.

Rigid ferrocenophane and its metal complexes with transition and alkaline-earth metal ions

The rigid [6]ferrocenophane, L-1, was synthesised by condensation of 1,1'-ferrocene dicarbaldehyde with trans-1,2-diaminocyclohexane in high dilution at r.t. followed by reduction. When other experimental conditions were employed, the [6,6,6]ferrocenephane (L-2) was also obtained. Both compounds were characterised by single crystal X-ray crystallography. The protonation of L-1 and its metal complexation were evaluated by the effect on the electron-transfer process of the ferrocene (fc) unit of L-1 using cyclic voltammetry (CV) and square wave voltammetry (SWV) in anhydrous CH3CN solution and in 0.1 M (Bu4NPF6)-Bu-n as the supporting electrolyte. The electrochemical process of L-1 between 300 and 900 mV is complicated by amine oxidation. On the other hand, an anodic shift from the fc/fc(+) wave of L-1 of 249, 225, 81 and 61 mV was observed by formation of Zn2+, Ni2+, Pd2+ and Cu2+ complexes, respectively. Whereas Mg2+ and Ca2+ only have with L-1 weak interactions and they promote the acid-base equilibrium of L-1. This reveals that L-1 is an interesting molecular redox sensor for detection of Zn2+ and Ni2+, although the kinetics of the Zn2+ complex formation is much faster than that of the Ni2+ one. The X-ray crystal structure of [(PdLCl2)-Cl-1] was determined and showed a square-planar environment with Pd(II) and Fe(II) centres separated by 3.781(1) angstrom. The experimental anodic shifts were elucidated by DFT calculations on the [(MLCl2)-Cl-1] series and they are related to the nature of the HOMO of these complexes and a four-electron, two-orbital interaction.

A Semiempirical Transition State Structure for the First Step in the Alkaline Hydrolysis of Cocaine. Comparison between the Transition State Structure, the Phosphonate Monoester Transition State Analog, and a Newly Designed Thiophosphonate Transition State Analog

Semiempirical molecular orbital calculations have been performed for the first step in the alkaline hydrolysis of the neutral benzoylester of cocaine. Successes, failures, and limitations of these calculations are reviewed. A PM3 calculated transition state structure is compared with the PM3 calculated structure for the hapten used to induce catalytic antibodies for the hydrolysis of cocaine. Implications of these calculations for the computer–aided design of transition state analogs for the induction of catalytic antibodies are discussed.

Effect of various alkaline-earth metal oxides on the broadband infrared luminescence from bismuth-doped silicate glasses

We report on the effect of various alkaline-earth metal oxides on the broadband infrared luminescence covering 1000-1600 nm wavelength region from bismuth-doped silicate glasses. The full width at half maximum (FWHM) of the infrared luminescence and the fluorescent lifetime is more than 200 nm and 400 mu s, respectively. The fluorescent intensity decreases with increasing basicity of host glasses. Besides the broadband infrared luminescence, luminescence centered at 640 nm was also observed, which should be ascribed to Bi2+ rather than to the familiar Bi3+. We suggest that the infrared luminescence should be assigned to the X-2 (2)Pi (3/2) -> X-1 (2)Pi(1/2) transition of BiO molecules dispersed in the host glasses. (c) 2006 Elsevier Ltd. All rights reserved.

AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg2+, Ca2+, Sr2+ and Ba2+ were used for,clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg2+, Ca2+ and Sr2+ are counterions. When Ba2+ is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr2+ induces the greatest structural transition.

Luminescence and its temperature effects on Sm2+ in alkaline earth borates

The luminescence of Sm2+ in alkaline earth berates (BaB8O13, SrB4O7 and SrB6O10) is reported. The temperature effects on luminescence and decay time of Sm2+ are studied. Due to the thermal population, D-5(1) --> F-7(J) transitions of Sm2+ in BaB8O13, SrB4O7 and SrB6O10 are observed at room temperature. The f-d broad emission transitions of Sm2+ in SrB4O7 and SrB6O10 are observed at high temperature whereas no f-d transition is observed in BaB8O13.

ANODIC-OXIDATION OF HYDRAZINE ON GLASSY-CARBON MODIFIED BY MACROCYCLIC TRANSITION-METAL COMPLEXES .1. COBALT PROTOPORPHYRIN DIMETHYL ESTER MODIFIED ELECTRODE

The electrocatalytic oxidation of hydrazine (N2H4) on a glassy carbon electrode (GC) modified by monolayer and polymer films of cobalt protoporphyrin dimethyl ester (CoPP) has been studied. Both the monolayer and polymer films of CoPP are very active to the anodic oxidation of N2H4. The activity of CoPP for the anodic oxidation of N2H4 is dependent on the pH of the solution, and the thickness of polymerized CoPP film. The oxidation kinetics were examined by methods of cyclic voltammetry, rotating disc electrodes and steady-state polarization measurement.

Kinetics and mechanism of a macrocyclic chromium(III) complex oxidation to chromium(IV) by hexacyanoferrate(III) in strongly alkaline media

Oxidation of the macrocyclic Cr(III) complex cis-[Cr(cycb)(OH)(2)](+), where cycb = rac-5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane, by an excess of the hexacyanoferrate( III) in basic solution, slowly produces Cr(V) species. These species, detected using e.p.r. spectroscopy, are stable under ambient conditions for many hours, and the hyperfine structure of the e.p.r. spectrum is consistent with the interaction of the d-electron with four equivalent nitrogen nuclei. Electro-spray ionization mass spectrometry suggests a concomitant oxidation of the macrocyclic ligand, in which double bonds and double bonded oxygen atoms have been introduced. By comparison basic chromate(III) solutions are oxidized rapidly to chromate(VI) by hexacyanoferrate(III) without any detectable generation of stable Cr(V) intermediates.

Alkaline hydrolysis of trinitrotoluene, TNT

The kinetics of the alkaline hydrolysis of trinitrotoluene, TNT, in an aqueous solution is a possible approach to destroying the active agent in unwanted munitions. The kinetics are shown to have a rapid initial step, step A, in which a highly coloured species, X (lambda(max) = 450 nm) is formed via an equilibrium reaction: TNT + OH- double left right arrow X. The bimolecular rate constant for the forward part of this equilibrium process, k(1), is: 0.099 +/- 0.004, 0.32 +/- 0.02 and 1.27 +/- 0.05 dm(3) mol(-1) s(-1), at 25, 40 and 60degreesC, respectively. The activation energy for the forward process is 60 kJ mol(-1). The first-order rate constant for the reverse of this process, k(-1), is: (5.3 +/- 2.6) x 10(-4), (1.2 +/- 1.0) x 10(-3) and (7.7 +/- 2.9) x 10(-3) s(-1) at 25, 40 and 60degreesC, respectively. The activation energy for the overall equilibrium process (k(1)/k(-1)) is ca. -5 kJ mol(-1). The subsequent alkaline hydrolysis of X to form the final product P, i.e. step B, is much slower than step A and appears to comprise two processes coupled in series, i.e. steps B1 (X +2OH(-) --> Z) and B2 (Z+OH- --> P). At 25degreesC, Step B1 appears rate determining throughout the decay process. At 45 degreesC and, more so, at 60degreesC, step B appears increasingly biphasic with increasing alkaline concentrations, as step B2 begins to compete with step B1 for position as the rate determining step. The trimolecular rate constant for step B1 is: 0.017 +/- 0.001, 0.0085 +/- 0.0002 and 0.0011 +/- 0.0001 dm(6) mol(-2) s(-1) at 25, 40 and 60degreesC, respectively, and the process has an activation energy of 64 kJ mol(-1). The transition from uniform kinetics, described by step B1, to mixed kinetics, described by steps B1 and B2, as the reaction temperature and alkali concentration are increased most likely occurs because (a) step B2 has a lower activation energy than B1, although it was not possible to measure the former parameter, and (b) step B2 has a lower (1st) order dependence upon [OH-] compared with that of step B1 (2nd). The bimolecular rate constant for step B2 is 0.0035 +/- 0.03 dm(3) mol(-1) s(-1) at 60degreesC. A brief NMR study of the initial hydrolysis product in water, acetone and chloroform, coupled with UV/visible spectra, provides evidence that species X is a Meisenheimer complex.

Determination of the nature of the diperiodatocuprate(III) species in aqueous alkaline medium through a kinetic and mechanistic study on the oxidation of iodide ion

The nature of the diperiodatocuprate(III) (DPC) species present in aqueous alkaline medium has been investigated by a kinetic and mechanistic study on the oxidation of iodide by DPC. The reaction kinetics were studied over the 1.0 ´ 10)3±0.1 mol dm)3 alkali range. The reaction order with respect to DPC, as well as iodide, was found to be unity when [DPC] [I)]. In the 1.0 ´ 10)3±1.0 ´ 10)2 mol dm)3 alkali region, the rate decreased with increase in the alkali concentration and a plot of the pseudo-®rst order rate constant, k versus 1/[OH)] was linear. Above 5.0 ´ 10)2 mol dm)3, a plot of k versus [OH)] was also linear with a non-zero intercept. An increase in ionic strength of the reaction mixtures showed no e ect on k at low alkali concentrations, whereas at high concentrations an increase in ionic strength leads to an increase in k. A plot of 1/k versus [periodate] was linear with an intercept in both alkali ranges. Iodine was found to accelerate the reaction at the three di erent alkali concentrations employed. The observed results indicated the following equilibria for DPC.

Crystal structures and thermal behavior of alkaline earth tricyanomethanides

The alkaline earth tricyanomethanides Mg(tcm)(2) center dot 2H(2)O, Ca(tcm)(2), Sr(tcm)(2) - H2O and Ba(tcm)(2) center dot 2H(2)O were prepared from aqueous solutions of the respective chlorides and silver tricyanomethanide. Their IR spectra and thermal behavior are described. The crystal structures of Ca(tcm)(2) and Ba(tcm)(2) center dot 2H(2)O were determined by single crystal X-ray diffraction. The structure of Ca(tcm)(2) is of the type found for several transition metal tricyanomethanides [1], containing two independent interpenetrating networks. Ba(tcm)(2) center dot 2H(2)O has a unique crystal structure corresponding to a three-dimensional coordination polymer with nine fold coordinated Ba atoms connected by water molecules and tricyanomethanide anions.