Catalytic currents of albumin at the hanging mercury drop electrode

Bovine serum albumin (BSA), as well as completely reduced BSA denoted by P (SH)₃₅, are adsorbed on the hanging mercury drop electrode (HMDE) from alkaline buffer solutions. When time is allowed, a monolayer is adsorbed from very dilute (10^?9M) BSA solutions in ammoniacal and borate buffers. With a monolayer of adsorbed protein the voltammograms at the HMDE are then identical in a given ammoniacal or borax buffer containing cobalt(III) or (II) and different BSA concentrations. Voltammograms of P (SH)₃₅ are virtually identical with those of native BSA. At the HMDE the second Brdi?ka current is proportional to concentration of cobalt(III) or (II) and the first current nearly so. Incompletely or completely adsorbed BSA or P (SH)₃₅ is not desorbed on keeping the HMDE for one hour in ammonia buffers. An incomplete layer of adsorbed BSA or P (SH)₃₅ is relatively rapidly desorbed at ?1.6 V (vs. SCE) and a complete film at ?1.65 V, some desorption occurring at ?1.6 V. Upon desorption, the second Brdi?ka current decreases faster than the first one; this is particularly striking in 1 M ammonia buffer. The rate of desorption is increased by calcium chloride, but the rate of adsorption is not, or only slightly, increased in the presence of calcium. Incomplete adsorption occurs at ?1.60 V (vs. SCE) and no adsorption at ?1.65 V. Indications are obtained that “presodium currents” yield a slight plateau at ?1.67 to ?1.70 V, the plateau currents being attributed to adsorbed BSA, while unadsorbed BSA yields catalytic currents without a plateau, the currents merging with the residual one of the buffer. Calcium chloride greatly increases the presodium currents. From many kinetic data obtained at the dropping mercury electrode (DME) and from results at the HMDE it is concluded that, depending on the BSA concentration, Brdi?ka currents at the DME are partly of a kinetic and partly of a surface adsorption nature and partly diffusion-controlled. Adsorption equilibrium is not attained at the DME at 25° at concentrations of BSA smaller than 10^?6M.     

Characteristics of a new catalytic hydrogen current observed with albumin in the presence of cobalt(III) or (II) at the hanging mercury drop electrode

When the hanging mercury drop electrode (HMDE) is placed in a solution which is 0.1 M in ammonia and 0.1 M in ammonium chloride and about 5 to 10×10^?4M in cobalt(III)-hexamine or cobalt(II) chloride and in very small concentrations of bovine serum albumin (BSA), the protein is slowly adsorbed. When the adsorption is highly incomplete and the HMDE is kept for 30 s at about ?1.05 V vs. SCE, “active cobalt’ is deposited as a complex (Co(0)BSA). This is anodically oxidized at about 0.0 V to unstable Co(I)BSA). When the electrode is then rapidly (500 mV s^?1) cathodized, a catalytic hydrogen current (i_c) with peak at circa ?1.45 V is observed. In this way it is even possible to detect and estimate BSA in concentrations of the order of 10^?12M. A detailed study has been made of the characteristics of i_c under several conditions. “Active cobalt” on the HMDE does not affect Brdi?ka currents. Cystine and cysteine also yield the catalytic hydrogen current i_c under the same conditions as does BSA.     

Cathodic stripping voltammetry of selenocystine,cystine, and cysteine in dilute aqueous acid

Selenocystine, cystine, and cysteine can be determined by cathodic stripping voltammetry (c.s.v.) in 0.1 M HClO₄ or 0.1 M H₂SO₄. The amino acids are accumulated at potentials more positive than —0.35 V, —0.20 V, and —0.10 V vs. s.c.e., respectively, and the stripping peak potentials are —0.45 V, —0.38 V and —0.15 V, respectively. Limiting coverage of the mercury electrode surface is observed for cysteine and selenocystine, but not for cystine. The detection limit for selenocystine is 5 × 10^-10 M in the presence of 100-fold amounts of cystine and cysteine. The detection limits for cystine and cysteine are 1 × 10^-5 M and 1 × 10^-9 M, respectively.     

Thermal Aggregation of Bovine Serum Albumin in Conventional Buffers: An Insight into Molecular Level Interactions

We have studied the effect of some conventional buffers, which are used frequently for biological research, on the thermal aggregation behavior of bovine serum albumin (BSA). The aggregation kinetics of BSA in buffer solutions were investigated by using UV–Vis spectroscopy. The buffers include sodium phosphate buffer, TRIS buffer and imidazole buffer at physiological pH (7.4). Dynamic light scattering and scanning electron microscopy have been employed to illustrate the size and morphology of protein aggregates. The molecular level interactions of buffer molecules with BSA was probed by various spectroscopic techniques, including UV–Vis, fluorescence, and circular dichroism. The results of this study reveal that the strong interactions of the buffers with protein’s folded/unfolded structures lead to stabilization/destabilization of BSA. We have also explored the possible binding sites of BSA for these buffers using a molecular docking technique.     

Electrochemical studies of homocysteine and homocystine at mercury electrodes

The behaviour of homocysteine and cysteine at mercury electrodes is compared. The one-electron oxidation associated with thiols is shown to be the same for both compounds in acidic phosphate buffer, giving rise to an adsorbed thiol—mercury complex, (RS)₂Hg, at the electrode surface. Formation of this complex is utilized in the cathodic stripping voltammetric determination of homocysteine; the detection limit is 10^?9 M after a deposition time of 90 s at a hanging mercury drop electrode. The similar E_{$\text{1}\text{2}$} values for homocysteine and cysteine mean that prior separation is needed for their individual determination. Amperometric detection with a mercury-coated goal electrode after separation by cation-exchange liquid chromatography provides a method for the simultaneous determination of both compounds. Reduction of homocystine at the mercury electrode is also compared to that of cystine. The more negative reduction potential, and the maximum observed for homocystine on d.c. polarograms, which is not seen for cystine, is attributable to different reaction kinetics at the mercury electrode; the products of both the 2-electron reductions are the corresponding thiol-containing amino acids.     

A study of the polarographic reduction of methaqualone

Methaqualone [2-methyl-3-o-tolyl-4(3H)-quinazolinone] is reduced at pH 1.5–5 in a single two-electron step. At pH 1.5–3, wave i₁ appears and is gradually replaced by wave i₂ which predomiantes in the solution between pH 4.5 and 6.5. At pH > 5, the height of wave i₂ decreases in the shape of a dissociaton curve with increasing pH. For both waves, the diprotonated form of methaqualone (I_a) is reduced to 1,2,-dihydromethaqualone as the final product. In wave i₁, the monoprotonated form I_b is further protonated at the electrode surface before it accepts the first electron; in wave i₂, the free base form I accepts two protons at the electrode surface before the first electron uptake. Polarographic curves are complicated by the presence of three waves of catalytic hydrogen evolution. Wave i_{1, cata} appears at pH < 5, waves i_2,cata at ?1.5 V and i_3,cata at ?1.7 V at pH > 5. Citrate buffers pH 1.5–3 or Britton-Robinson buffers at pH 2.6–3.6 are most suitable for quantitative work with eitehr d.c. polarography or differential pulse polarography.     

Chemical forms of cobalt(0) related to polarogrphic and voltammetric catalytic hydrogen currents

The mechanism of catalytic hydrogen evolution at a mercury electrode in buffer solutions containing both bovine serum albumin (BSA) and Co(II) or Co(III) is discussed on the basis of the amount of Co(0) formed on the electrode as well as the solubility of the Co(0) species. An appropriate potential was times t, in ammonia buffer solutions in the presence of Co(III). After the mercury drop was washed with 0.1 M ammonia buffer (pH 9.3) and 1 M hydrochloric acid or concentrated hot hydrochloric acid, the amount of cobalt remaining in and/or on the mercury drop was determined by flameless atomic absorption spectrometry. Amalgamation of Co(0), which is insoluble in the ammonia buffer and 1 M hydrochloric acid, was formed on the electrode, regardless of the electrolysis potential, when t was short. With increase of t, Co(0) changed its characteristics, yielding a Co(0) species [Co(0)_{Group 2}] soluble in 1 M hydrochloric acid but insoluble in the buffer, even though the time required for the change depended on the electrolysis potential. It is demonstrated that Co(0)_{Group 2} plays an important role in the appearance of such catalytic hydrogen currents as the first and second Brdička currents, the P-current of Anzenbacher and Kalous, and i_c of Kolthoff and Kihara.     

Determination of cystine and cysteine in seawater using cathodic stripping voltammetry in the presence of Cu(II)

A method is described for the determination of cystine and cysteine in seawater and freshwater using cathodic stripping voltammetry in the presence of added copper(II). The optimized conditions include a copper concentration of 150 nM, a pH of 8.5, and a collection potential of −0.15 V; the cathodic reduction peak is located at −0.55 V. The detection limit is 0.1 nM after a collection period of 4 min. The sensitivity is diminished by surfactants similar to Triton X-100 in natural waters; the sensitivity therefore needs to be calibrated by internal standard additions of amino acids. It is possible to differentiate between cystine and cysteine by employing a solution pH of 6.2, where the peak due to cystine is absent. The response in seawater is different from that previously reported in buffer solutions. It is shown that the amino acid reduction peak is due to Cu(I) reduction of the adsorbed Cu(I)-cysteinate complex; this complex is formed with Cu(I) generated from dissolved copper(II) at the electrode surface at −0.15 V in the presence of cysteine; cystine is reduced to cysteine at the electrode surface during the collection process.     

Simple, Rapid, and Accurate RP-HPLC Method for Determination of Cystine in Human Urine after Derivatization with Dansyl Chloride

An accurate, simple, and sensitive reversed-phase high-performance liquid chromatographic method, with loratadine as internal standard (IS) and UV detection at 286 nm, has been developed for deterination of cystine in human urine. The major innovations of the method include use of acrylonitrile to protect cysteine from oxidization to cystine, separation of cysteine, as the dansyl derivative, from cystine, and use of isocratic elution instead of gradient elution to reduce the time and cost of serial analysis. The mobile phase was 0.05 M sodium acetate–methanol, 35:65 (v/v), adjusted to pH 3.5 with 2.5 M citric acid, at a flow rate of 1.0 mL min^?1. The retention times of cystine and the IS were 16.6 and 19.9 min, respectively. The limit of detection for cystine was 0.3 mg L^?1. Extraction recovery of cystine was >85.6%. Intra-day and inter-day precision (RSD) for cystine were below 4.3 and 8.5%, respectively. There was no chromatographic interference from other α-amino acids present in mammalian proteins, or from other urine components. The calibration plot for the cystine derivative was linear in the range 1–500 mg L^?1 and the correlation coefficient was 0.9992. The method was validated appropriately and successfully used for determination of cystine in human urine.     

Formation of azatitanacyclopentanes from ene-imines and a Ti(O-i-Pr)4/2i-PrMgX reagent and their synthetic reactions

ω-Vinylimines reacted with a Ti(O-i-Pr)₄/2i-PrMgX reagent to generate the corresponding azatitanacyclopentanes in quantitative yield, which in turn reacted with H₂O, I₂ and O₂ to give 2-methyl-, 2-iodomethyl-, 2-hydroxymethyl-1-aminocyclic compounds, respectively. The azatitanacyclopentanes thus generated reacted with formaldehyde to afford the corresponding 2,3-annulated pyrrolidines in good yield.     

Dosage du platine par spectrométrie d'absorption atomique: Partie II. Résultats analytiques sur les complexes or-ganométalliques d'intérêt biologique: effet cis-trans

An atomic-absorption spectrometric method is reported for the determination of platinum in a series of platinum complexes, with and without the use of spectrochemical buffers. The use of potassium phosphate and lanthanum chloride as buffer eliminated interference effects. The results were reproducible and accurate Platinum phosphine complexes were easily analysed. The cis and trans PtL₂L^'₂ (L,L^' are ligands) complexes soluble in water gave significant differences in absorbance in the absence of spectrochemical buffers, so that absorbance measurements differentiated cis- and trans-compounds in a given series. The cis-trans effect was also detected in organic solvents.     

Spectrophotometric determination of formaldehyde with chromotropic acid in phosphoric acid medium assisted by microwave oven

In the present study, a spectrophotometric method for the determination of formaldehyde by using chromotropic acid was devised, in which the use of potentially hazardous and corrosive concentrated sulfuric acid was eliminated and advantageously replaced by a mixture of H₃PO₄ and H₂O₂. The reaction between formaldehyde and chromotropic acid (CA) in a concentrated phosphoric acid medium was accelerate by irradiating the mixture with microwave energy for 35 s (1100 W), producing a violet-red compound (λ_max=570 nm). Beer's Law is obeyed in a concentration range of 0.8-4.8 mg l⁻¹ of formaldehyde with a good correlation coefficient (r=0.9968). The proposed method was applied in the analysis of formaldehyde in commercial disinfectants. Recoveries were within 98.0-100.4%, with standard deviations ranging from 0.03 to 0.13%.     

Phytochemical mediated gold nanoparticles and their PTP 1B inhibitory activity

Current discovery demonstrates the rapid formation of gold nanoparticles with guavanoic acid a phytochemical of Psidium guajava (Pg). The pharmacological capabilities of the phytochemicals present in the leaves of Pg and their ability to generate gold nanoparticles is presented herein. The new genre of green nanoparticles exhibit remarkable Protein Tyrosine Phosphatase 1B (PTP 1B) inhibitory activity and in vitro stability in various physiological medium including saline, histidine, cysteine, bovine serum albumin (BSA), human serum albumin (HSA) and buffers (pH 5, 7 and 9). It is predicted that this new technology will be felt greatly in several routes of pharmaceuticals.     

A contribution to the voltammetric study of cystine and cysteine at Pt electrodes in 0.5 M H2SO4

Both cysteine and cystine adsorb at the Pt electrode according the Frumkin—Temkin isotherm with the heterogeneity factor f = 51 for cysteine and 21 for cystine. Both the adsorbed cysteine and cystine give in a solution without any dissolved cystine or cysteine almost identical first cyclic voltammetric curves. Each substance dissolved in the electrolyte gives two oxidation peaks which differ when the oxidation is carried out at a “reduced” or an “oxidized” Pt electrode. On the basis of the dependence of the height and potential of the peaks on polarization rate and concentration (in the case of oxidation of dissolved substances) and of coulometric measurements the following conclusions have been made concerning the kinetics and mechanism:(i) Neither cysteine nor cystine change their oxidation state on adsorption at the electrode.(ii) The final oxidation product of both adsorbed cysteine and cystine may be the cysteic acid.(iii) For cysteine there are two adsorbed species, one strongly adsorbed, the other one weakly adsorbed.(iv) The oxidation of dissolved cysteine takes place via the weakly adsorbed species, the surface concentration of which is influenced by the coverage of the strongly adsorbed species. This process is described by an electrode reaction rate equation.(v) In the overall oxidation of cysteine one electron is transferred while the detailed mechanism requires an oxidation by splitting-off two electrons with a subsequent ion—substrate dimerization reaction.     

Accurately measuring respiratory activity of single living cells by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) is a powerful tool to examine the respiratory activity of living cells. However, in SECM measurements of cell respiratory activity, the signal recorded usually also includes the signal corresponding to the cell topography. Therefore, measurements of cell respiratory activity using conventional SECM techniques are not accurate. In the present work, we develop a method for accurate measurement of the respiratory activity of single living cells using SECM. First, cells are immobilized on a glass substrate modified with collagen. Then, a Pt ultramicroelectrode tip of SECM held at −0.50 V is scanned along the central line across a living cell and a SECM scan curve, i.e., the relationship of the tip current versus the displacement (the first scan curve) is recorded with a negative peak. The peak current i_p on this first scan curve is composed of i_p1, which corresponds to the cell respiratory activity and i_p2, which corresponds to the cell topography. In order to isolate the i_p2 component, the cell is killed by exposing it to 1.0 × 10⁻³ mol/L KCN for 10 min. The tip is then scanned again with the same trace over the dead cell, and a second SECM scan curve is recorded. Noting that the topography of the dead cell is the same as that of the living cell, this second scan curve with a negative peak corresponds now only to the cell topography. Thus, i_p2 is obtained from the second SECM scan curve. Finally, i_p1 corresponding to the respiratory activity of the living cell can be accurately calculated using i_p1 = i_p − i_p2. This method can be used to monitor real-time change in the respiratory activity of single cells after exposing them to KBr, NaN₃ and KCN.     

Development of a rapid and sensitive method for determination of cysteine/cystine ratio in chemically defined media

Two rapid, sensitive and quantitative methods for the determination of the cysteine and cystine ratio in complex defined media feedstock using monolithic reversed-phase liquid chromatography (RPLC) and RPLC–MS are presented. Cysteine is pre-derivatised with purified 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) and separated from other derivatisation products on a narrow-bore 50 mm × 2 mm I.D. monolithic C₁₈ column with UV detection at 355 nm. For reversed-phase LC (RPLC) the separation is carried out isocratically using a mobile phase of 50 mM trichloroacetic acid (TCA) adjusted to pH 2.5 with lithium hydroxide (LiOH) and acetonitrile (83:14) pumped at 1.5 mL/min with an elevated column temperature. For RPLC–MS an ammonium acetate and acetonitrile gradient method was developed with a reduced flow rate of 0.3 mL/min. The treatment of the samples consisted of dividing them into two aliquots, the first aliquot is analysed for cysteine and the second aliquot is analysed for cystine after its quantitative reduction to cysteine using tris(2-carboxyethyl)phosphine (TCEP). Both methods are linear, with R² > 0.999 for 0.25–500 μM for cysteine and 0.25–250 μM for cystine using the LC–UV method, sensitive, with detection limit of 36 nM for cysteine, and precise, with ≤1.1% RSD for both retention time and peak area (n = 6). Samples (n = 31) of an industry standard and supplied chemically defined media feedstock were analysed, finding cysteine ranging from 1.56 to 2.26 μg/mL and cystine from 1062.02 to 1348.13 μg/mL.     

Voltammetric study on interaction of cysteine with type I-collagen in the aqueous medium

Interaction of cysteine with type I-collagen from bovine achilles tendon in the aqueous solutions has been examined using square wave voltammetry (SWV) and cyclic voltammetry (CV) techniques. In the absence of cysteine, type I-collagen gives a reversible peak at ?0.114 V in Britton-Robinson (B-R) buffer (pH 4.0). The electrochemical parameters (I _p/f, E _p/f, E _p/pH, I _p/pH, I _p/v, I _p/v^1/2) of type I-collagen have been also studied. In addition, it has been determined that there is a linear relationship between current and concentration of type I-collagen. On the other hand, cysteine exhibits a reversible peak at ?0.383 V due to the reduction of mercurous cysteine thiolate. By using a hanging mercury drop electrode in aqueous solutions, SWV and CV voltammograms obtained for type I-collagen in the presence of cysteine indicate that there is an interaction between type I-collagen and cysteine. In the presence of cysteine, peak current of type I-collagen decreases and a new peak is observed at ?0.121 V for cysteine which is bonded to type I-collagen.     

Cu(II) and Pd(II) complexes of N-(2-hydroxybenzyl)aminopyridines

N-(2-Hydroxybenzyl)aminopyridines (Lⁱ) react with Cu(II) and Pd(II) ions to form complexes in the compositions Cu(Lⁱ)₂(CH₃COO)₂ · nH₂O (n = 0, 2, 4), Pd(Lⁱ)₂Cl₂ · nC₂H₅OH (n = 0, 2) and Pd(L²)₂Cl₂ · 2H₂O. In the complexes, the ligands are neutral and monodentate which coordinate through pyridinic nitrogen. Crystal data of the complexes obtained from 2-amino pyridine derivative have pointed such a coordinating route and comparison of the spectral data suggests the validity of similar complexation modes of other analog ligands. Cu(II) complex of N-(2-hydroxybenzyl)-2-aminopyridine (L¹), [Cu(L¹)₂(CH₃COO)₂] has slightly distorted square planar cis-mononuclear structure which is built by two oxygen atoms of two monodentate carboxylic groups disposed in cis-position and two nitrogen atoms of two pyridine rings. The remaining two oxygen atoms of two carboxylic groups form two Cu and H bridges containing cycles which joint at same four coordinated copper(II) ion. IR and electronic spectral data and the magnetic moments as well as the thermogravimetric analyses also specify on mononuclear octahedric structure of complexes [Cu(L²)₂(CH₃COO)₂ · 2H₂O] and [Cu(L³)₂(CH₃COO)₂ · 4H₂O] where L² and L³ are N-(2-hydroxybenzyl)-2- or 3-aminopyridines, respectively.     

Pulse polarography: Part X. Formaldehyde hydration in aqueous acetate and phosphate buffer solutions

Formaldehyde hydration has been studied with pulse polarography in acetate and phosphate buffers, at 25°C. It could be shown that in both buffer systems extrapolation to zero buffer concentration produces the equilibrium constant K_d=[CH₂O]/[CH₂(OH)₂]=(6.31±0.05) 10^?4 in pure water, a value of as yet unmatched precision. In the buffers mentioned two novel features appear in the hydration mechanism: (a) buffer acids form addition compounds with CH₂O where the, corresponding equilibria are rapidly established. The equilibrium constants have been derived. (b) H₂PO₄^?/HPO₄^2? buffers are exceptional in that the polarographic specific rate of hydration as a function of the concentration of this buffer is less than expected on the basis of a normal linear dependence. This is explained, as in the case of free-aldehyde glucose [23], on the basis of a solvent (water)-shared associate of (presumably) H₂PO₄^? with CH₂O which decreases the rate of diffusion of the latter, the more so as the buffer concentration increases. It is pointed out, that the occurrence of such associates can explain the excess activity of H₂PO₄^? as an acid catalyst in aldehyde hydration [16].     

Chiral hydroxythiols as catalysts for enantioselective borane ketone reductions

Several chiral 1,2- and 1,3-hydroxythiols derived from (R)-camphor, (1S)-(+)-10-camphorsulfonyl chloride, cysteine and cystine derivatives were prepared and evaluated as catalysts in borane ketone reductions. Under the best experimental conditions (10 mol% catalyst, THF, 35°C), a 95% yield of (R)-1-phenylethanol of 64% ee was obtained in the borane reduction of acetophenone.