Determination of ranitidine in urine by capillary electrophoresis-electrochemiluminescent detection

The fast analysis of ranitidine is of clinical importance in understanding its efficiency and a patient's treatment history. In this paper, a novel determination method for ranitidine based on capillary electrophoresis-electrochemiluminescence detection is described. The conditions affecting separation and detection were investigated in detail. End-column detection of ranitidine in 5 mM Ru(bpy)(3)(2+) solution at applied voltage of 1.20 V was performed. Favorable ECL intensity with higher column efficiency was achieved by electrokinetic injection for 10s at 10 kV. The R.S.D. values of ECL intensity and migration time were 6.38 and 1.84% for 10(-4) M and 6.01 and 0.60% for 10(-5) M, respectively. A detection limit of 7 x 10(-8) M (S/N=3) was achieved. The proposed method was applied satisfactorily to the determination of ranitidine in urine in 6 min.     

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.     

Determination of ascorbic acid in individual rat hepatocyte by capillary electrophoresis with electrochemical detection

A method for the direct determination of ascorbic acid (AA) in individual rat hepatocyte based on capillary electrophoresis (CE) coupled with electrochemical detection (ECD) using a new kind of homemade carbon fiber micro-disk bundle electrode has been described. Individual rat hepatocytes were injected into a fused-silica capillary with an inner diameter of 25 microm, and lysed by 0.1% sodium dodecylsulfate (SDS) as cell lysis solution. The following conditions were suitable for the determination of AA: running buffer, 1.83 x 10(-2) mol/l Na2HPO4-1.70 x 10(-3) mol/l NaH2PO4 (pH 7.8); separation voltage, 20.0 kV; detection potential, 0.80 V (vs. saturated calomel electrode (SCE)). The concentration limit of detection (LOD) of the method was 1.7 x 10(-6) mol/l at a signal-to-noise (S/N) ratio of 3, and the mass LOD was 3.0 fmol. The linear dynamic range was from 5.0 x 10(-6) to 5.0 x 10(-4) mol/l with a correlation coefficient of 0.9962 for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the peak current. This method was successfully applied to AA determination in rat hepatocyte. The recovery was between 91% and 97%, and the amount of AA in single rat hepatocyte ranged from 28 to 63 fmol.     

Methanol plug assisted sweeping-micellar electrokinetic chromatography for the determination of dopamine in urine by violet light emitting diode-induced fluorescence detection

The use and limitations of a methanol plug assisted sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method is described. Using naphthalene-2,3-dicarboxaldehyde (NDA)-labeled dopamine as a model compound, this new method was also used in the determination of dopamine in actual urine samples. An inexpensive violet light emitting diode (LED) was used for the light source, because this is suitable for fluorescence excitation. The number of theoretical plates of the analyte was determined to be approximately 1 x 10(5) and approximately 2 x 10(5) by means of MEKC and sweeping-MEKC and this was improved to approximately 1 x 10(6) when the methanol plug assisted mode was applied. In addition, the detection limit of NDA-labeled dopamine was determined to be 9.1 x 10(-7) and 1.2 x 10(-8)M by means of MEKC and sweeping-MEKC and this was improved to 4.7 x 10(-9)M when the methanol plug assisted sweeping-MEKC mode was applied.     

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.     

Simple determination of trace amounts of anionic surfactants in river water by spectrophotometry combined with solid-phase extraction

A simple and sensitive spectrophotometric method combined with solid-phase extraction (SPE) for the simultaneous determination of sodium linear-dodecylbenzenesulfonate (DBS) and sodium dodecyl sulfate (SDS) is described. The C2 (ethyl group bonded silicagel) cartridge could be repeatedly used more than 500 times for SPE, and it enabled the anionic surfactants to be concentrated by 50-fold. The calibration graph for DBS was linear in the range from 1.6 x 10(-8) M to 5.0 x 10(-7) M and for SDS from 2.0 x 10(-9) M to 3.0 x 10(-7) M. The relative standard deviation (n=5) for 5.0 x 10(-7) M DBS was 3.1% and for 2.5 x 10(-7) M SDS was 1.7%. The proposed method was applied to the simultaneous determination of DBS and SDS in river-water samples.     

High-performance liquid chromatographic determination of pyrophosphate in the presence of a 20,000-fold excess of orthophosphate.

An HPLC method was based on anion-exchange separation of pyrophosphate (diphosphate) and orthophosphate and postcolumn spectrophotometric detection at 140 degrees C with a molybdenum(V)-molybdenum(VI) reagent. The reagent was easy to prepare, stable for at least 6 months at room temperature, and ready for the determination of pyrophosphate and orthophosphate by the so-called heteropoly blue method without use of any reducing agent. A photodiode-array detector for HPLC indicated the spectral characteristics of the heteropoply blue complex that was detectable at 330-800 nm. The HPLC method had a wide dynamic range from 3 x 10(-7) to 5 x 10(-4) M for both pyrophosphate and orthophosphate with a relative standard deviation of measurement of 10 approximately 2%. Pyrophosphate of 5 x 10(-7) and 5 x 10(-6) M, respectively, could be determined in the presence of a 20,000-fold excess of orthophosphate; 0.01 and 0.1 M.     

Aryl hydrocarbon receptor response to indigoids in vitro and in vivo

Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC50 of approximately 5x10(-6)M (indirubin 3' -oxime EC50 approximately 5x10(-7)M, indirubin EC50 approximately 1x10(-7)M). A comparison of these EC50 values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) ( approximately 3x10(-9)M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC50 values for indirubin and TCDBF ( approximately 2x10(-9) and 5x10(-9)M), with the IC50 values of indigo and indirubin 3' -oxime being approximately 10-fold higher. When rats were treated with these indigoids in the range of 1.5-50mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.     

The genomes of Desulfovibrio gigas and D. vulgaris

Two-dimensional electrophoresis of sequential double-restriction digests showed that the genome of Desulfovibrio gigas compromised 1.63 x 10(6) bp (1.09 x 10(9) Dal) of DNA; an ammonia-limited chemostat population possessed an average of nine genomes per cell and a multiplying batch culture possessed approximately 17 genomes per cell. The genome size of D. vulgaris (Hildenborough) was 1.72 x 10(6) bp (1.14 x 10(9) Dal); a population from an ammonia-limited batch culture contained four genomes per cell. Control digestions and analyses with Escherichia coli GM4 agreed reasonably with published values: a genome size of 3.95 x 10(6) bp and approximately two genomes per cell from a stationary batch culture in glucose minimal medium. Desulfovibrio gigas carried two plasmids of approximately 70 MDal (1.05 x 10(5) bp) and approximately 40 MDal (6 x 10(4) bp); D. vulgaris (Hildenborough) contained one of approximately 130 MDal (1.95 x 10(5) bp). Single plasmids were also detected in a second strain of D. vulgaris and in strain Berre sol of D. desulfuricans but not in 10 other desulfovibrios including representatives of D. desulfuricans, D. vulgaris, D. salexigens and D. africanus.     

Studies on the mechanism of oxidative phosphorylation. ADP promotion of GDP phosphorylation

The process of ATP or GTP synthesis by bovine heart submitochondrial particles involves the binding of ADP or GDP to 3 exchangeable sites I, II, and III, and only upon substrate occupation of site III does rapid ATP or GTP synthesis take place. The dissociation constants determined for ADP were KADPI less than or equal to 10(-8) M, KADPII approximately 10(-7) M, and KADPIII (equivalent to apparent KADPm), approximately 3 x 10(-6) M in the low Km mode and KADPIII approximately 150 x 10(-6) M in the high Km mode. For GDP, these constants were KGDPI approximately 10(-6)-10(-5) M, KGDPII approximately 10(-4) M, and KGDPIII approximately 10(-3) M when NADH was the respiratory substrate (Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Biol. Chem. 265, 82-88). Because of its low affinity for the above binding sites, GDP at micromolar concentrations does not lead to GTP synthesis. However, as shown in this paper, micromolar [GDP] undergoes phosphorylation in the presence of micromolar concentrations of ADP. Under these conditions, both ATP and GTP are synthesized. GDP inhibits ATP synthesis with KGDPi congruent to 7 microM, while ADP promotes GTP synthesis in a reaction that requires inorganic phosphate (apparent KPim = 2-3 mM) and is inhibited by uncouplers and inhibitors of the ATP synthase complex. The ADP-promoted GTP synthesis exhibited an "apparent" KGDPm = 4 microM and an "apparent" Vmax = 11 nmol of GTP (min.mg of protein)-1. These results were interpreted to mean that (a) micromolar [ADP] occupies sites I and II, allowing site III to bind and phosphorylate GDP, and (b) the KGDPm and Vmax calculated under these conditions represent values for the low Km-low Vmax mode of GTP synthesis, which in the absence of ADP is not detectable because of the positive cooperativity phase of GTP synthesis with the high KGDPII approximately 10(-4) M.     

Engineering the microstructure and permeability of thin multilayer latex biocatalytic coatings containing E. coli.

The microstructure and permeability of rehydrated 20-100 microm thick partially coalesced (vinyl-actetate acrylic copolymer) SF091 latex coatings and a 118 microm thick model trilayer biocatalytic coating consisting of two sealant SF091 layers containing a middle layer of viable E. coli HB101 + latex were studied as delaminated films in a diffusion apparatus with KNO(3) as the diffussant. The permeability of the hydrated coatings is due to diffusive transport through the pore space between the partially coalesced SF091 latex particles. Coating microstructure was visualized by fast freeze cryogenic scanning electron microscopy (cryo-SEM). The effective diffusion coefficient of SF091 latex coatings (diffusive permeability/film thickness) was determined as the ratio of the effective diffusivity of KNO(3) to its diffusivity in water (D(eff)/D). Polymer particle coalescence was arrested by two methods to increase coating permeability. The first used glycerol with coating drying at 4 degrees C, near the glass transition temperature (T(g)). The second method used sucrose or trehalose as a filler to arrest coalescence; the filler was then dissolved away. D(eff)/D was measured as a function of film thickness; content of glycerol, sucrose, and trehalose; drying time; and rehydration time. D(eff)/D varied from 3 x 10(-4) for unmodified SF091 coatings to 6.8 x 10(-2) for coatings containing sucrose. D(eff)/D was reduced by the flattening of latex particles against the surface of the solid substrate, as well as by the presence of the colloid stabilizer hydroxyethylcellulose (HEC). When corrected for the flattened particle layer, D(eff)/D of HEC-free coatings was as high as 0.20, which agreed with the value predicted from analysis of cryo-SEM images of the coat surface. D(eff)/D decreased by one-half in approximately 5 days in rehydrated SF091 coatings, indicating that significant wet coalescence occurs after glycerol, sucrose, or trehalose are leached from the films. D(eff)/D of SF091 latex trilayer coatings containing viable E. coli HB101 cells decreased as cell loading was increased from 2.2 x 10(-2) for 64 g dry cell weight per liter of coat volume to 5 x 10(-3) for 151 g DCW/L of coat volume. The reduction in coating permeability with increasing cell loading is predicted by Maxwell's equation for D(eff)/D in periodic composites.     

A new film for the fabrication of an unmediated H2O2 biosensor

A novel and stable film made from polyethylene glycol (PEG) on pyrolytic graphite (PG) electrode was presented in this paper for incorporating horseradish peroxidase (HRP) to study the direct electrochemistry of the enzyme. In PEG film, HRP showed a thin-layer electrochemistry behavior. The apparent standard potential (E degrees ') was -0.379 V versus SCE at pH 7.2. Moreover, the PEG-HRP modified electrode exhibited excellent electrocatalytical response to the reduction of H2O2 with a calibration range between 2.0 x 10(-6) and 6.0 x 10(-4) M and a good linear relation from 2.0 x 10(-6) to 1.0 x 10(-4) M, on which an unmediated H2O2 biosensor was based. The detection limit of 6.7 x 10(-7) M was estimated when the signal-to-noise ratio was 3. The relative standard deviation (R.S.D.) was 4.7% for six successive determinations at a concentration of 4.0 x 10(-5) M. The apparent Michaelis-Menten constant (Km app) of the sensor was found to be 1.38 mM. Epinephrine, dopamine, and ascorbic acid did not interfere with the sensitive determination of H2O2.     

Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.     

Kinetics and thermodynamics of lipid amphiphile exchange between lipoproteins and albumin in serum

We have examined the kinetics and thermodynamics of the exchange of a fluorescent amphiphile derived from a phospholipid, NBD-DMPE, between serum albumin and the serum lipoproteins of high density (HDL2 and HDL3), LDL, and VLDL. Binding of the fluorescent lipid amphiphile to bovine serum albumin is characterized, at 35 degrees C, by an equilibrium binding constant of approximately 3 x 10(6) M(-1) and a characteristic time < or = 0.1 s. Association of NBD-DMPE with the lipoprotein particles, if considered as a partitioning of amphiphile monomers between the aqueous phase and the lipoprotein particles, is characterized by an equilibrium partition coefficient between 10(5) and 10(6), being highest for LDL and lowest for HDL. The association of NBD-DMPE monomers with lipoprotein particles can be described by insertion rate constants on the order of 10(5) M(-1) s(-1) for VLDL and LDL and 10(4) M(-1) s(-1) for HDL. The desorption rate constants are on the order of 10(-5) s(-1) for all particles. The study was performed as a function of temperature between 15 and 35 degrees C. This permitted the calculation of the equilibrium thermodynamic parameters (deltaG(o), deltaH(o), and deltaS(o)) as well as the activation parameters (deltaG++(o), deltaH++(o), and deltaS++(o)) for the insertion and desorption processes. The association equilibrium is dominated by the entropic contribution to the free energy in all cases. The results are discussed in relation to phospholipid and amphiphile exchange phenomena involving the lipoproteins.     

Nuclear oestrogen receptors in rat liver. Development of assay conditions, characterization and the translocation of oestrogens in vivo.

A method for the determination of specific oestrogen-receptor binding sites in rat liver nuclei is described. Nuclear receptors showed a high affinity for oestradiol (Kd approximately 3 x 10(-9)M), a low capacity, and a distinct specificity for substances with known oestrogenic and anti-oestrogenic activity. No sex differences were seen in the concentrations of nuclear receptors from either vehicle- or ethynyloestradiol-pretreated rats. Only a limited number of binding sites could be extracted with 0.4 M-KCl. The remaining sites, which were solubilized by sonication and treatment with deoxyribonuclease I, sedimented at 3-4 S. Of four oestrogens tested (oestradiol, ethynyloestradiol, diethylstilboestrol, tri-p-anisylchloroethylene), ethynyloestradiol was the most effective translocation agent in vivo, nuclear uptake occurring at doses below 1 microgram/rat; changes in salt extractability of nuclear receptors occurred at doses lower than those required to achieve absolute increases in nuclear receptor concentrations.     

Advantages of CCD detectors for de novo three-dimensional structure determination in single-particle electron microscopy

For three-dimensional (3D) structure determination of large macromolecular complexes, single-particle electron cryomicroscopy is considered the method of choice. Within this field, structure determination de novo, as opposed to refinement of known structures, still presents a major challenge, especially for macromolecules without point-group symmetry. This is primarily because of technical issues: one of these is poor image contrast, and another is the often low particle concentration and sample heterogeneity imposed by the practical limits of biochemical purification. In this work, we tested a state-of-the art 4 k x 4 k charge-coupled device (CCD) detector (TVIPS TemCam-F415) to see whether or not it can contribute to improving the image features that are especially important for structure determination de novo. The present study is therefore focused on a comparison of film and CCD detector in the acquisition of images in the low-to-medium ( approximately 10-25 A) resolution range using a 200 kV electron microscope equipped with field emission gun. For comparison, biological specimens and radiation-insensitive carbon layers were imaged under various conditions to test the image phase transmission, spatial signal-to-noise ratio, visual image quality and power-spectral signal decay for the complete image-processing chain. At all settings of the camera, the phase transmission and spectral signal-to-noise ratio were significantly better on CCD than on film in the low-to-medium resolution range. Thus, the number of particle images needed for initial structure determination is reduced and the overall quality of the initial computed 3D models is improved. However, at high resolution, film is still significantly better than the CCD camera: without binning of the CCD camera and at a magnification of 70 kx, film is better beyond 21 A resolution. With 4-fold binning of the CCD camera and at very high magnification (> 300 kx) film is still superior beyond 7 A resolution.     

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex

The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT > TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.     

An enzymatic determination of hydrogen peroxide by using spectrophotometry.

A new spectrophotometric method for determining low hydrogen peroxide concentrations by using horseradish peroxidase in the presence of NADH at pH 7.5 has been described. Both total NADH consumption and initial reaction rate may be used for the determination. Using the NADH consumption, a linear response with respect to hydrogen peroxide was observed in the concentration range 7 x 10(-8)-2.5 x 10(-6) M. Due to the presence of superoxide dismutase, hydrogen peroxide is partly regenerated and an amplification of the signal results, which explains the sensitivity.     

Protonation dynamics of the alpha-toxin ion channel from spectral analysis of pH-dependent current fluctuations.

To probe protonation dynamics inside the fully open alpha-toxin ion channel, we measured the pH-dependent fluctuations in its current. In the presence of 1 M NaCl dissolved in H2O and positive applied potentials (from the side of protein addition), the low frequency noise exhibited a single well defined peak between pH 4.5 and 7.5. A simple model in which the current is assumed to change by equal amounts upon the reversible protonation of each of N identical ionizable residues inside the channel describes the data well. These results, and the frequency dependence of the spectral density at higher frequencies, allow us to evaluate the effective pK = 5.5, as well as the rate constants for the reversible protonation reactions: kon = 8 x 10(9) M-1 s-1 and koff = 2.5 x 10(4) s-1. The estimate of kon is only slightly less than the diffusion-limited values measured by others for protonation reactions for free carboxyl or imidazole residues. Substitution of H2O by D2O caused a 3.8-fold decrease in the dissociation rate constant and shifted the pK to 6.0. The decrease in the ionization rate constants caused by H2O/D2O substitution permitted the reliable measurement of the characteristic relaxation time over a wide range of D+ concentrations and voltages. The dependence of the relaxation time on D+ concentration strongly supports the first order reaction model. The voltage dependence of the low frequency spectral density suggests that the protonation dynamics are virtually insensitive to the applied potential while the rate-limiting barriers for NaCl transport are voltage dependent. The number of ionizable residues deduced from experiments in H2O (N = 4.2) and D2O (N = 4.1) is in good agreement.     

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.