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Kinetic Methods (kinetic + methods)

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Chemistry	80%

Selected Abstracts

The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73

FEBS JOURNAL, Issue 22 2002
Alona Pavlova
The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by ,,300-fold, >10-fold and ,,4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by ,,10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one amino-acid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, ,,45% of the total binding energy, for inhibition of cathepsin B. [source]

Oxidation of diclofenac sodium by diperiodatoargantate(III) in aqueous alkaline medium and its determination in urine and blood by kinetic methods

INTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 6 2010
P. N. Naik
The kinetics and oxidation of diclofenac sodium (DFS) by diperiodatoargentate(III) (DPA) in alkaline medium at 298 K and at a constant ionic strength of 0.60 mol dm,3 were studied spectrophotometrically. The oxidation products were [2-(2,6-dicloro-phynylamino)-phenyl]-methenol and Ag(I), identified by LC-ESI-MS and IR spectral studies. The reaction between DFS and DPA in alkaline medium exhibits 1:1 stoichiometry. The reaction is first order in [DPA] and has a less than unit order dependence each in [DFS] and [alkali]. Increasing concentrations of IO,4 retard the reaction. The active species of DPA proposed to be monoperiodatoargentate(III), and a mechanism is suggested. The rate constants involved in the different steps of the mechanism were determined and are discussed. The activation parameters with respect to a rate-limiting step of the mechanism were determined. The thermodynamic quantities were also determined. Using the oxidation of DFS by DPA, DFS was analyzed by kinetic methods in urine and blood sample. The proposed method enables DFS analysis in the range from 5.0 × 10,5 to 5.0 × 10,3 mol dm,3. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 336,346, 2010 [source]

Pharmacokinetic,pharmacodynamic integration of orbifloxacin in rabbits after intravenous, subcutaneous and intramuscular administration

JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2008
P. MARÍN
The single-dose disposition kinetics of orbifloxacin were determined in clinically normal rabbits (n = 6) after intravenous (i.v.), subcutaneous (s.c.) and intramuscular (i.m.) administration of 5 mg/kg bodyweight. Orbifloxacin concentrations were determined by high performance liquid chromatography with fluorescence detection. Minimal inhibitory concentrations (MICs) assay of orbifloxacin against 30 strains of Staphylococcus aureus from several European countries was performed in order to compute pharmacodynamic surrogate markers. The concentration,time data were analysed by compartmental and noncompartmental kinetic methods. Steady-state volume of distribution (Vss) and total body clearance (Cl) of orbifloxacin after i.v. administration were estimated to be 1.71 ± 0.38 L/kg and 0.91 ± 0.20 L/h·kg, respectively. Following s.c. and i.m. administration orbifloxacin achieved maximum plasma concentrations of 2.95 ± 0.82 and 3.24 ± 1.33 mg/L at 0.67 ± 0.20 and 0.65 ± 0.12 h, respectively. The absolute bio-availabilities after s.c. and i.m. routes were 110.67 ± 11.02% and 109.87 ± 8.36%, respectively. Orbifloxacin showed a favourable pharmacokinetic profile in rabbits. However, on account of the low AUC/MIC and Cmax/MIC indices obtained, its use by i.m. and s.c. routes against the S. aureus strains assayed in this study cannot be recommended given the risk of selection of resistant populations. [source]

Kinetic, Thermodynamic, and Mechanistic Patterns for Free (Unbound) Cytochrome c at Au/SAM Junctions: Impact of Electronic Coupling, Hydrostatic Pressure, and Stabilizing/Denaturing Additives

CHEMISTRY - A EUROPEAN JOURNAL, Issue 27 2006
Dimitri E. Khoshtariya Prof. Dr.
Abstract Combined kinetic (electrochemical) and thermodynamic (calorimetric) investigations were performed for an unbound (intact native-like) cytochrome c (CytC) freely diffusing to and from gold electrodes modified by hydroxyl-terminated self-assembled monolayer films (SAMs), under a unique broad range of experimental conditions. Our approach included: 1) fine-tuning of the charge-transfer (CT) distance by using the extended set of Au-deposited hydroxyl-terminated alkanethiol SAMs [-S-(CH2)n -OH] of variable thickness (n=2, 3, 4, 6, 11); 2) application of a high-pressure (up to 150,MPa) kinetic strategy toward the representative Au/SAM/CytC assemblies (n=3, 4, 6); 3) complementary electrochemical and microcalorimetric studies on the impact of some stabilizing and denaturing additives. We report for the first time a mechanistic changeover detected for "free" CytC by three independent kinetic methods, manifested through 1) the abrupt change in the dependence of the shape of the electron exchange standard rate constant (ko) versus the SAM thickness (resulting in a variation of estimated actual CT range within ca. 15 to 25 Å including ca. 11 Å of an "effective" heme-to-,-hydroxyl distance). The corresponding values of the electronic coupling matrix element vary within the range from ca. 3 to 0.02 cm,1; 2) the change in activation volume from +6.7 (n=3), to ,0 (n=4), and ,5.5 (n=6) cm3,mol,1 (disclosing at n=3 a direct pressure effect on the protein's internal viscosity); 3) a "full" Kramers-type viscosity dependence for ko at n=2 and 3 (demonstrating control of an intraglobular friction through the external dynamic properties), and its gradual transformation to the viscosity independent (nonadiabatic) regime at n=6 and 11. Multilateral cross-testing of "free" CytC in a native-like, glucose-stabilized and urea-destabilized (molten-globule-like) states revealed novel intrinsic links between local/global structural and functional characteristics. Importantly, our results on the high-pressure and solution-viscosity effects, together with matching literature data, strongly support the concept of "dynamic slaving", which implies that fluctuations involving "small" solution components control the proteins' intrinsic dynamics and function in a highly cooperative manner as far as CT processes under adiabatic conditions are concerned. [source]