Peroxidase-like activities of iron(III)—Porphyrins: kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by anilines

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10⁵ M^?1 sec^?1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10⁵ M^?1 sec^?1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions (k_dfh)found to be related to those for the enzymic reactions (k_hrp) by the equation:log k_DFH= 0.65log k_HRP+ 1.96 with a correlation coefficient of 0. 98.     

Reactions of iron-sulfur proteins with the aquated electron

The reaction of parsley 2Fe-2S ferredoxin in the normal oxidized state with e_aq^? generated by pulse radiolysis techniques has been studied at ～25°C, pH 7–8, I = 0.10 M (NaClO₄). Rate constants k_e (e_aq^? decay) and k_p (protein absorbance change) are the same, second-order rate constant 9.7 × 10⁹ M^?1 sec^?1. The reaction exhibits close to 100% efficiency. With 8Fe-8S ferredoxin from Clostridium pasteurianum under identical conditions it now appears that k_p (although sometimes significantly smaller) is equal to k_e. Varying efficiencies are also observed with this protein depending on the batch used. The reasons for such variable behavior are not fully understood. With oxidized and reduced forms of Chromatium v. high-potential iron-sulfur protein (HIPIP), k_e and k_p are essentially the same, but the highest efficiency observed is only ～50%. The prevailing pattern is therefore that rate constants k_e and k_p are generally in step for proteins having a single (or identical) active site(s). When the active site is buried as with HIPIP the efficiency of the reaction appears to decrease.     

Nonenzymatic reduction of nitrosobenzene to phenylhydroxylamine by NAD(P)H

The nonenzymatic reduction of nitrosobenzene by NADPH and NADH in aqueous buffer solution at 25°C is described. Both reactants quantitatively convert nitrosobenzene to phenylhydroxylamine. Rate constants for reduction (k_r) were determined spectrophotometrically and found to be identical at pH 5.7 and 7.4 and independent of buffer concentration. The values of k_NADH (124–149 M^?1 sec^?1) and k_NADPH (131–170 M^?1 sec^?1) are essentially identical. The reaction is not subject to general catalysis or specific salt effects. The oxidation of phenylhydroxylamine by NAD(P) to nitrosobenzene is only stimulated by a factor of 1.2 over oxidation in its absence (when the ratio of NADP: phenylhydroxylamine was 8:1).     

Solvent Effect on the Photolysis of Riboflavin

The kinetics of photolysis of riboflavin (RF) in water (pH 7.0) and in organic solvents (acetonitrile, methanol, ethanol, 1-propanol, 1-butanol, ethyl acetate) has been studied using a multicomponent spectrometric method for the assay of RF and its major photoproducts, formylmethylflavin and lumichrome. The apparent first-order rate constants (k_obs) for the reaction range from 3.19 (ethyl acetate) to 4.61 × 10⁻³ min⁻¹ (water). The values of k_obs have been found to be a linear function of solvent dielectric constant implying the participation of a dipolar intermediate along the reaction pathway. The degradation of this intermediate is promoted by the polarity of the medium. This indicates a greater stabilization of the excited-triplet states of RF with an increase in solvent polarity to facilitate its reduction. The rate constants for the reaction show a linear relation with the solvent acceptor number indicating the degree of solute–solvent interaction in different solvents. It would depend on the electron-donating capacity of RF molecule in organic solvents. The values of k_obs are inversely proportional to the viscosity of the medium as a result of diffusion-controlled processes.KEY WORDS: dielectric constant, kinetics, photolysis, riboflavin, solvent effect, viscosity     

Kinetic data for redox reactions of cytochrome c with Fe(CN)5X complexes and the question of association prior to electron transfer

Use of rigorous equilibration kinetics to evaluate rate constants for the Fe(CN)6 4- reduction of horse-heart cytochrome c in the oxidized form, cyt c (III), has shown that limiting kinetics do not apply with concentrations of Fe(CN)6 4- (the reactant in excess) in the range 2-10 x 10(-4) M, I = 0.10 M (NaCl). The reaction conforms to a first-order rate law in each reactant, and at 25 degrees C, pH 7.2 (Tris), it is concluded that K for association prior to electron transfer is less than 200 M-1. From previous studies at 25 degrees C, ph 7.0 (10(-1) M phosphate), I = 0.242 M (NaCl), a value K = 2.4 x 10(3) M-1 has been reported. Had such a value applied, some or all of the redox inactive complexes Mo(CN)8 4-, Co(CN)6 3-, Cr(CN)6 3-, Zr(C2O4)4 4- present in amounts 5-20 x 10(-4) M would have been expected to associate at the same site and partially block the redox process. No effect on rats was observed. With the reductants Fe(CN)5(4-NH2-py)3- and Fe(CN)5(imid)3-, reactions proceeded to greater than 90% completion and rate laws were again first order in each reactant. Rate constants (M-1 sec-1) at 25 degrees C, pH 7.2 (Tris), I = 0.10 M (NaCl), are Fe(CN)6 4- (3.5 x 10(4)), Fe(CN)5(4-NH2py)3- (6.7 x 10(5), and Fe(CN)5(imid)3- (4.2 x 10(5). Related reactions in which cyt c(II) is oxidized are also first order in each reactant, Fe(CN)6 3- (9.1 x 10(6)), Fe(CN)5(NCS)3- (1.3 x 10(6)), Fe(CN)5(4-NH2py)2- (3.8 x 10(6) at pH 9.4), and Fe(CN)5(NH3)2- (2.75 x 10(6) at ph 8). Redox inactive Co(CN)6 3- (1.0 x 10(-3) M) has no effect on the reaction of Fe(CN)6 3- which suggests that a recent interpretation for the Fe(CN)6 3- oxidation of cyt c(II), I = 0.07 M, may also require reappraisal.     

NAD-phenol complex formation, the inhibition of malate dehydrogenase by phenols, and the influence of phenol substitutents on inhibitory effectiveness.

Kinetic analyses indicate that inhibition by phenols of the forward reaction of malate dehydrogenase involves the binding of two molecules of phenol. One is bound as phenol, the other as a charge transfer complex of phenol with NAD. Inhibition of the reverse reaction by phenol involves the binding of only a single phenol molecule per active unit of enzyme. Kinetic evidence for this binding pattern is supported by spectral evidence in which ultraviolet absorbance and circular dichroism studies show binding of the NAD-phenol complex by malate dehydrogenase. Circular dichroism difference spectra indicate that phenol alone also binds to malate dehydrogenase.The apparent inhibition constants for fourteen variously substituted phenols were found to be significantly correlated with the hydrophobic binding constant (π), the Hammet σ function and the NAD-phenol charge transfer association constant of the individual phenols. The degree of dependency of the apparent K_i on the hydrophobicity of phenols suggests that the observed inhibition occurs via binding of phenol and/or NAD-phenol complex in hydrophobic regions of the malate dehydrogenase molecule.     

Phenol derivatives as enhancers and inhibitors of luminol–H2O2–horseradish peroxidase chemiluminescence

Systematic studies on phenol derivatives facilitates an explanation of the enhancement or inhibition of the luminol–H₂O₂–horseradish peroxidase system chemiluminescence. Factors that govern the enhancement are the one-electron reduction potentials of the phenoxy radicals (PhO^•/PhOH) vs. luminol radicals (L^•/LH⁻) and the reaction rates of the phenol derivatives with the compounds of horseradish peroxidase (HRP-I and HRP-II). Only compounds with radicals with a similar or greater reduction potential than luminol at pH 8.5 (0.8 V) can act as enhancers. Radicals with reduction potentials lower than luminol behave in a different way, because they destroy luminol radicals and inhibit chemiluminescence. The relations between the reduction potential, reaction rates and the Hammett constant of the substituent in a phenol suggest that 4-substituted phenols with Hammett constants (σ) for their substituents similar or greater than 0.20 are enhancers of the luminol–H₂O₂–horseradish peroxidase chemiluminescence. In contrast, those phenols substituted in position 4 for substituents with Hammett constants (σ) lower than 0.20 are inhibitors of chemiluminescence. On the basis of these studies, the structure of possible new enhancers was predicted. © 1998 John Wiley & Sons, Ltd.     

Inhibition of cobalt(II) carboxypeptidase A by ligands. Kinetic evidence for the formation of a ternary enzyme-metal-ligand complex

Saturation kinetics are observed in the inhibition of cobalt carboxypeptidase A by the chelating agent 1,10-phenanthroline. The association constant K₁ for the formation of the enzyme-metal-ligand ternary complex and k₂, the rate of breakup of the ternary complex, have been obtained. A mechanism is proposed to account for the pH profile of the reaction which, in conjunction with K₁, permits the calculation of the individual rate constants k₁, K_?1, k₂, k₃. The magnitude of the rate constant k₁ suggests that cobalt(II) in CoCPA is five-coordinate. Similar but less extensive studies on inhibition by 2,2′-bipyridyl and 8-hydroquinoline-5-sulfonic acid have also been carried out     

Computer calculation-based quantitative structure-activity relationships for the oxidation of phenol derivatives horseradish peroxidase compound II

 The second-order rate constants for the oxidation of a series of phenol derivatives by horseradish peroxidase compound II were compared to computer-calculated chemical parameters characteristic for this reaction step. The phenol derivatives studied were phenol, 4-chlorophenol, 3-hydroxyphenol, 3-methylphenol, 4-methylphenol, 4-hydroxybenzoate, 4-methoxyphenol and 4-hydroxybenzaldehyde. Assuming a reaction of the phenolic substrates in their non-dissociated, uncharged forms, clear correlations (r = 0.977 and r = 0.905) were obtained between the natural logarithm of the second-order rate constants (ln k _app and ln k ₂ respectively) for their oxidation by compound II and their calculated ionisation potential, i.e. minus the energy of their highest occupied molecular orbital [E(HOMO)]. In addition to this first approach in which the quantitative structure-activity relationship (QSAR) was based on a calculated frontier orbital parameter of the substrate, in a second and third approach the relative heat of formation (ΔΔHF) calculated for the process of one-electron abstraction and H^• abstraction from the phenol derivatives was used as a parameter. Plots of the natural logarithms of the second-order rate constants (k _app and k ₂) for the reaction and the calculated ΔΔHF values for the process of one-electron abstraction also provide clear QSARs with correlation coefficients of –0.968 and –0.926 respectively. Plots of the natural logarithms of the second-order rate constants (k _app and k ₂) for the reaction and the calculated ΔΔHF values for the process of H^• abstraction provide QSARs with correlation coefficients of –0.989 and –0.922 respectively. Since both mechanisms considered, i.e. initial electron abstraction versus initial H^• abstraction, provided clear QSARs, the results could not be used to discriminate between these two possible mechanisms for phenol oxidation by horseradish peroxidase compound II. The computer calculation-based QSARs thus obtained for the oxidation of the various phenol derivatives by compound II from horseradish peroxidase indicate the validity of the approaches investigated, i.e. both the frontier orbital approach and the approach in which the process is described by calculated relative heats of formation. The results also indicate that outcomes from computer calculations on relatively unrelated phenol derivatives can be reliably compared to one another. Furthermore, as the actual oxidation of peroxidase substrates by compound II is known to be the rate-limiting step in the overall catalysis by horseradish peroxidase, the QSARs of the present study may have implications for the differences in the overall rate of substrate oxidation of the phenol derivatives by horseradish peroxidase. Received: 29 March 1996 / Accepted: 17 July 1996     

Kinetics of cyanide binding to chloroperoxidase in the presence of nitrate: detection of the influence of a heme-linked acid group by shift in the appa

The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant (K_CN) of the peroxidase-cyanide complex and both forward (k₊) and reverse (k_?) rate constants are independent of the H⁺ concentration over the pH range 2.7 to 7.1. The values obtained are k_cn = (9.5 ± 1.0) × 10^-5 M, k₊. = (5.2 ± 0.5) × 10⁴ M^?1 sec^?1 and k_- = (5.0± 1.4) sec^-1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher pK_a values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the pK_a of the heme-linked acid group.     

Merged mechanisms for hydride transfer from 1,4-dihydronicotinamides

Recent work on the reduction of heteroaromatic cations by 1,4-dihydronicotinamides and related reducing agents is reviewed. Extensive correlations are presented between the second-order rate constants (k₂) for these reactions and the second-order rate constants (k_OH) and equilibrium constants (pK_R⁺) for hydroxide ion attack on these cations. Close correlations of log k₂ with the electron affinities and one-electron reduction potentials of these cations are also presented. These relationships are considered in the context of a direct hydride transfer from donor to acceptor and also in terms of SET mechanisms which are also commonly discussed for such reactions. It is shown that the interpretation of these formal hydride transfer reactions in terms of an imbalanced development of electronic charge and C---H bond fission within the transition state species leads to a rational merging of the single-step hydride transfer mechanism and the SET mechanisms. The structures of the transition state species are expected to be highly variable and quite dependent upon the nature of the hydride donor and acceptor species, with considerable contribution from charge-transfer interactions. Such imbalanced transition state species are analyzed in terms of two different types of reaction coordinate diagrams and also in terms of the valence bond configuration mixing theory.     

In vitro characterization of the Ac locus in white clover (Trifolium repens L.)

Microsomal fractions from developing shoots of adult white clover plants (of genotype AcAc) and cotyledons of dark germinated clover seedlings can synthesize 2-hydroxy-2-methylpropanenitrile and 2-hydroxy-2-methylbutanenitrile, the aglycone precursors of the cyanogenic glucosides, linamarin and lotaustralin, from various precursors in the presence of NADPH. l-Valine, 2-methylpropanal oxime, and 2-methylpropanenitrile are converted to 2-hydroxy-2-methylpropanenitrile and are detected as cyanide and acetone. l-Isoleucine and 2-methylbutanal oxime are converted to 2-hydroxy-2-methylbutanenitrile and are detected as cyanide and 2-butanone. At least two steps in these conversions are missing in microsomes from plants of genotype acac.     

Lanthanide ion catalysis of the trans-cis isomerization of trans-bis(oxalato)-diaquochromate(III) and trans-bis(malonato)diaquochromate(III) [1]

The lanthanide ion catalyzed trans-cis isomerizations of trans-bis(oxalato)diaquochromate(II) and trans-bis(malonato)diaquochromate(III) have been studied. A linear free energy relationship was found correlating the catalytic rate constants for the oxalate reaction with the corresponding formation constants of complexes formed between simple monocarboxylic acids and the light (LaGd) members of the lanthanide series. The results indicates that for this portion of the series, the reaction mechanism is related to the formation of monocarboxylate complex intermediates. When the ionic radius of the lanthanide ion decreases below a particular value (as in the latter half of the series), the metal ion remains coordinated to both carboxylates of the oxalate ion rather than simply binding to only one carboxylate. In either situation, isomerization to the cis product eventually occurs, and the lanthanide ion is released.The reaction rates associated with the trans-bis(malonato)diaquochromate(III) reaction were found to be significantly slower than those of the corresponding oxalate system. However, in the malonate system, no linear free energy relationship was found relating the catalytic rate constants with the corresponding formation constants of monocarboxylic acids. One does find a linear relationship between the catalytic rate constants for the malonate reaction and the log K₁ values for the corresponding lanthanide/malonate complexes. During the course of the trans-cis isomerization, the lanthanide ion chelates the dissociated malonate group of a pentavalent Cr(III) intermediate. In the mechanism the lanthanide ion does not aid in ring opening, and neither does it singly bond to the intermediate     

Biotransformation of benzene and toluene to catechols by phenol hydroxylase from Arthrobacter sp. W1

Phenol hydroxylase gene engineered microorganism (PH_IND) was used to synthesize catechols from benzene and toluene by successive hydroxylation reaction. HPLC-MS and ¹H NMR analysis proved that the products of biotransformation were the corresponding catechols via the intermediate production of phenols. It was indicated that the main products of toluene oxidation were o-cresol and p-cresol. 3-Methylcatechol was the predominant product for m-cresol biotransformation. Formation rate of catechol (25 μM/min/g cell dry weight) was 1.43-fold higher than that of methylcatechols. It was suggested that phenol hydroxylase could be successfully used to transform both benzene and toluene to catechols by successive hydroxylation.     

Microbial Transformation of Esters of Chlorinated Carboxylic Acids

Two groups of compounds were selected for microbial transformation studies. In the first group were carboxylic acid esters having a fixed aromatic moiety and an increasing length of the alkyl component. Ethyl esters of chlorine-substituted carboxylic acids were in the second group. Microorganisms from environmental waters and a pure culture of Pseudomonas putida U were used. The bacterial populations were monitored by plate counts, and disappearance of the parent compound was followed by gas-liquid chromatography as a function of time. The products of microbial hydrolysis were the respective carboxylic acids. Octanol-water partition coefficients (K_ow) for the compounds were measured. These values spanned three orders of magnitude, whereas microbial transformation rate constants (k_b) varied only 50-fold. The microbial rate constants of the carboxylic acid esters with a fixed aromatic moiety increased with an increasing length of alkyl substituents. The regression coefficient for the linear relationships between log k_b and log K_ow was high for group 1 compounds, indicating that these parameters correlated well. The regression coefficient for the linear relationships for group 2 compounds, however, was low, indicating that these parameters correlated poorly.     

Accumulation and excretion of pesticides used as insecticides or fungicides in agricultural products by the willow shiner Gnathopogon caerulescens

The average bioconcentration factors (BCF) in the whole body of willow shiner (Gnathopogon caerulescens) after 24–336 hr exposure were 810 for chlorpyriphosmethyl, 802 for vamidothion, 110 for edifenphos, 25 for ethoprophos, 83 for bendocarb, 39 for pirimicarb and 114 for methyl parathion. The correlations between a logarithm of 48 hr-Lc₅₀ to carp (log Lc₅₀ and the parameters (a logarithm of n-octanol-water partition coefficiens (log P_ow) and log BCF in willow shiner) were investigated for the pesticides studied here and already reported. The correlation factor (r) was −0.136 (N = 21) for log Lc₅₀ vs log P_ow and 0.039 (N = 24) for log Lc ₅₀ vs log BCF. The excretion rate constants (k) from the whole body of the fish were 0.01 hr⁻¹ for chlorpyriphosmethyl, 0.03 hr⁻¹ for vamidothion, 0.11 hr⁻¹ for edifenphos, 0.27 hr⁻¹ for ethoprophos, 0.18 hr⁻¹ for bendiocarb, 0.01 hr⁻¹ for pirimicarb and 0.08 hr⁻¹ for methyl parathion. The correlation between log k and log BCF was investigated for 22 pesticides already reported and studied here. The r value was not so high (−0.537, N = 22) but higher (−0.672, N = 2) in the case of excluding simetryne.     

Borate buffer inhibition of peroxidatic intermediate formation in the deutero-ferriheme-hydrogen peroxide system

The rate of formation of peroxidatically active reaction intermediate(s) via oxidation of the iron(III)-porphyrin complex, deuteroferriheme, with hydrogen peroxide decreases with increasing borate content of mixed borate-carbonate buffer solutions. Studies at pH = 9.25 in 0.035 M borate buffer and 0.035 M carbonate buffer suggest borate to function as an uncompetitive inhibitor. A comparison of slopes and intercepts of double reciprocal plots for inhibited and uninhbited reactions allows calculation of selected parameters for the deuteroferriheme-H₂O₂ reaction at pH = 9.25 in terms of a typical enzymatic stoichiometric mechanism for heme activity. This includes the Michaelis constant (K_m = 8.1 × 10^?5 M) and the first-order rate constant for conversion of heme-substrate complex to intermediate(s) (k₃ = 7.4 sec^?1). A tentative mechanistic model involving reversible interaction of borate inhibitor with heme-substrate complex is considered, and pseudo-first-order rate constants calculated on the basis of this scheme are in reasonable agreement with those obtained experimentally. It is suggested that comparable inhibitory action may be responsible for some previously reported cases of decreased catalase enzyme activity in borate buffer solutions     

Real-time Monitoring of Intermediates Reveals the Reaction Pathway in the Thiol-Disulfide Exchange between Disulfide Bond Formation Protein A (DsbA) and B (DsbB) on a Membrane-immobilized Quartz Crystal Microbalance (QCM) System

Disulfide bond formation protein B (DsbB_S-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbA_SH,SH). The oxidized DsbA_S-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbA_SH,SH and DsbB_S-S,S-S. We immobilized DsbB_S-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (k_f, k_r, and k_cat, respectively) indicated that the two pairs of cysteine residues in DsbB_S-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbB_S-S,S-S. Our data suggested that the reaction pathway of almost all DsbA_SH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.     

Proton uptake mechanism of bacteriorhodopsin as determined by time-resolved stroboscopic-FTIR-spectroscopy

Bacteriorhodopsin's proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 μs, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k₇ = 0.3 ms, k₄ = 2.3 ms, k₃ = 6.9 ms, k₆ = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k₄), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M₁ to M₂ transition. However, experimentally two different M intermediates are not resolved, and M₂ and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M₂) transition. This reaction is proposed to be involved in the “reset switch” of the pump, (b) In the M to N (conceptionally M₂) transition, most likely, asp-85's carbonyl frequency shifts from 1,762 to 1,753 cm^-1 and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O.     

Influence of membrane structure on the kinetics of carrier-mediated ion transport through lipid bilayers

Charge-pulse relaxation experiments of valinomycin-mediated Rb⁺ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experimental data the rate constants of association (k_R) and dissociation (k_D) of the ion-carrier complex as well as the rate constants of translocation of the complex (k_MS) and of the free carrier (k_S) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants k_S and k_MS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C₂₀ fatty acid from one to four resulted in an increase of k_S by a factor of seven and in an increase of k_MS by a factor of twenty-four. The stability constant K = k_R/k_D of the ion-carrier complex as well as the translocation rate constants k_S and k_MS were found to depend strongly on the nature of the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced k_R, k_MS and k_S up to seven-fold.