Simultaneous determination of phenylethanoid glycosides and aglycones by capillary zone electrophoresis with running buffer modifier

Although the separation efficiency of capillary electrophoresis (CE) is much higher than that of other chromatographic methods, it is sometimes difficult to adequately separate the complex ingredients in biological samples. This article describes how one effective and simple way to develop the separation efficiency in CE is to add some modifiers to the running buffer. The suitable running buffer modifier β-cyclodextrin (β-CD) was explored to fast and completely separate four phenylethanoid glycosides and aglycones (homovanillyl alcohol, hydroxytyrosol, 3,4-dimethoxycinnamic acid, and caffeic acid) in Lamiophlomis rotata (Lr) and Cistanche by capillary zone electrophoresis with ultraviolet (UV) detection. It was found that when β-CD was used as running buffer modifier, a baseline separation of the four analytes could be accomplished in less than 20 min and the detection limits were as low as 10⁻³ mg L⁻¹. Other factors affecting the CE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage, and sample injection time, were investigated extensively. Under the optimal conditions, a successful practical application on the determination of Lr and Cistanche samples confirmed the validity and practicability of this method.     

On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10⁻⁸–1.0 × 10⁻⁶ M with a detection limit of 1.0 × 10⁻⁸ M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10⁻⁸, 5.0 × 10⁻⁷ and 1.0 × 10⁻⁶ M MTX (n = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases

A simple and fast dynamically coated capillary electrophoretic method was developed for the characterization and inhibition studies of alkaline phosphatases (EC 3.1.3.1). An inside capillary enzymatic reaction was performed, and hydrolysis of the substrate 4-nitrophenylphosphate to 4-nitrophenol was measured. Fused-silica capillary surface was dynamically modified with polycationic polybrene coating. By reversal of the electroosmotic flow (EOF), analysis time was reduced up to 3 min as the anionic analytes were migrated in the same direction as the EOF. Furthermore, the sensitivity of the method was increased using electroinjection through high-field amplified injection. The baseline separation of 4-nitrophenylphosphate and 4-nitrophenol was achieved by employing 50 mM sodium phosphate as the running buffer (pH 8.5), 0.0025% polybrene, and a constant voltage of −15 kV, and the products were detected at 322 nm. Under the optimized conditions, a good separation with high efficiency was achieved. The new method was applied to study enzyme kinetics and inhibitor screening. K_m and K_i values obtained with the new CE method were compared well with the standard spectrophotometric method. Dynamic coating of fused-silica capillary gave fast and reproducible separation of substrate and product. The method can be easily optimized for inhibition studies of other isozymes.     

A capillary electrophoresis-based enzyme assay for kinetics and inhibition studies of carbonic anhydrase

In the current study, capillary electrophoresis (CE)-based enzyme assay for characterization and inhibition study of bovine carbonic anhydrase II (bCA II) was developed. The developed method is the first CE assay for carbonic anhydrase (CA). The method was optimized in order to get short analysis time, minimal sample volume consumption, and high resolution of substrate and product. The CE conditions were optimized as follows: fused-silica capillary (30 cm effective length × 75 μm i.d.), pressure injection for 5 s, 20 mM sodium borate buffer (pH 9.0), constant voltage of 15 kV, constant capillary temperature of 25 °C, and detection at 260 nm. For precise measurements, uridine was used as an internal standard during optimization of the CE methods. The limits of detection and quantification for p-nitrophenyl acetate (p-NPA) were 3.01 and 9.12 μM, respectively, whereas for p-nitrophenolate they were 2.05 and 6.22 μM, respectively. The performance of the developed method was confirmed by determination of kinetic parameters (i.e., K_m and V_max of bCA for p-NPA); the inhibition constant (K_i) was determined for furosemide, a standard inhibitor of CA. The new method proved to be fast and efficient, and it can be used for the investigation of inhibitors of all isoforms of CAs.     

A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase

Protoporphyrinogen oxidase (PPO) is a flavin adenine dinucleotide (FAD)-containing enzyme in the tetrapyrrole biosynthetic pathway that leads to the formation of both heme and chlorophylls, which has been identified as one of the most important action targets of commercial herbicides. The literature reports gave different PPO-catalytic kinetic parameters for the substrate protoporphyrinogen IX (K_m of 0.1 to 10.4 μM) with different sources of PPO using fluorescent or HPLC methods. Herein we assayed the enzymatic activity of recombinant Bacillus subtilis PPO by using capillary electrophoresis (CE), a method with high separation efficiency, easy automation, and low sample consumption. The Michaelis constant and maximum reaction velocity were determined as 7.0 ± 0.6 μM and 0.38 ± 0.02 μmol min^-1 μg⁻¹, respectively. The interaction between PPO and acifluorfen, a commercial PPO-inhibiting herbicide, was measured as the inhibition constant 186.9 ± 9.3 μМ. The relationship between cofactor FAD and PPO activity can also be quantitatively studied by this CE method. The CE method used here should also be a convenient, reliable method for PPO study.     

Interaction of ruthenium(III) chloride with bis(3,5-dimethylpyrazol-1-yl)methane oxyanions, potential κ-N,N,O scorpionates

When RuCl₃ was set to react with both bis(3,5-dimethylpyrazol-1-yl)acetate (bdmpza) and bis(3,5-dimethylpyrazol-1-yl)methane sulfonate (bdmpzsa) new ruthenium(II) complexes were obtained. The reduction of ruthenium(III) was studied by the NMR Evans method and spectrophotometrically, for 1:1 (Ru:L) molar ratios. Using the Evans method pseudo first-order constants of 2.5 × 10⁻³ s⁻¹ (bdmpzsa) and 3.9 × 10⁻³ s⁻¹ (bdmpza) were obtained in DMSO-d₆ (2% t-butanol) solutions. Spectrophotometrically the corresponding constants were also calculated: 1.1 × 10⁻³ s⁻¹ for bdmpzsa, and 1.6 × 10⁻³ s⁻¹, for bdmpza. Both ligands behave as κ³-N,N,O scorpionates but with a weak oxyanionic coordination to the metal, susceptible to be substituted with NEt₃ for a 1:1 molar ratio.     

Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection

The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB-PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE-mass spectrometry (MS) of this sample, using a PB-PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE-UV and CE-MS indicated the presence of both deamidation and oxidation products.     

A chemically modified lipase preparation for catalyzing the transesterification reaction in even highly polar organic solvents

Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It was found that 1,2-dimethoxyethane was the best precipitant which precipitated 97% protein and complete activity. PCMC (protein coated microcrystals), CLPCMC (crosslinked protein coated microcrystals), EPROS (enzyme precipitated and rinsed with organic solvents) and pH tuned preparations of modified and unmodified lipase were prepared and used for carrying out transesterification reaction with n-octane and dimethyl formamide (DMF) as reaction medium. In n-octane, among all the preparations, CLPCMC of modified lipase gave highest rate (1970 nmol min⁻¹ mg⁻¹) as compared to unmodified pH tuned lipase (128 nmol min⁻¹ mg⁻¹). In DMF, with both 1% (v/v) and 5% (v/v) water content, CLPCMC showed highest initial rate of 0.72 and 7.2 nmol min⁻¹ mg⁻¹, respectively. Unmodified pH tuned lipase showed no activity at all in DMF with both 1% and 5% (v/v) water content.     

Large-volume sample stacking of selected drugs of forensic significance by capillary electrophoresis

Large-volume sample stacking capillary electrophoresis (LVSS-CE) and conventional capillary electrophoresis (CE) are compared for the separation of drugs of significance to forensic and clinical analyses. LVSS-CE for cations requires the use of an electroosmotic flow (EOF) modifier in conjunction with polarity switching to effect on-column concentration of an analyte and its subsequent migration in the capillary. The run buffer consists of 0.05 mol dm⁻³ disodium tetraborate adjusted to pH 2.2 with orthophosphoric acid, and the EOF modifier is 0.002 mol dm⁻³ cetyltrimethylammonium bromide. CE investigations used an identical run buffer minus the EOF modifier. LVSS-CE and CE investigations used injection times of 30 s and 3 s, respectively. Both modes of capillary electrophoresis are compared in terms of their limits of detection, efficiency, resolution and reproducibility. LVSS-CE is also applied to the analysis of a spiked urine sample.     

Nafion/lead nitroprusside nanoparticles modified carbon ceramic electrode as a novel amperometric sensor for l-cysteine

This work describes the electrochemical and electrocatalytic properties of carbon ceramic electrode (CCE) modified with lead nitroprusside (PbNP) nanoparticles as a new electrocatalyst material. The structure of deposited film on the CCE was characterized by energy dispersive X-ray (EDX), Fourier transform infrared (FTIR), and scanning electron microscopy (SEM). The cyclic voltammogram (CV) of the PbNP modified CCE showed two well-defined redox couples due to [Fe(CN)₅NO]³⁻/[Fe(CN)₅NO]²⁻ and Pb^IV/Pb^II redox reactions. The modified electrode showed electrocatalytic activity toward the oxidation of l-cysteine and was used as an amperometric sensor. Also, to reduce the fouling effect of l-cysteine and its oxidation products on the modified electrode, a thin film of Nafion was coated on the electrode surface. The sensor response was linearly changed with l-cysteine concentration in the range of 1 × 10⁻⁶ to 6.72 × 10⁻⁵ mol L⁻¹ with a detection limit (signal/noise ratio [S/N] = 3) of 0.46 μM. The sensor sensitivity was 0.17 μA (μM)⁻¹, and some important advantages such as simple preparation, fast response, good stability, interference-free signals, antifouling properties, and reproducibility of the sensor for amperometric determination of l-cysteine were achieved.     

Mechanism and biological implications of the NO release of cis-[Ru(bpy)2L(NO)](n+) complexes: a key role of physiological thiols

Nitric oxide (NO) has a critical role in several physiological and pathophysiological processes. In this paper, the reactions of the nitrosyl complexes of [Ru(bpy)₂L(NO)]ⁿ⁺ type, where L = SO₃²⁻ and imidazole and bpy = 2,2′-bipiridine, with cysteine and glutathione were studied. The reactions with cysteine and glutathione occurred through the formation of two sequential intermediates, previously described elsewhere, [Ru(bpy)₂L(NOSR)]ⁿ⁺ and [Ru(bpy)₂L(NOSR)₂] (SR = thiol) leading to the final products [Ru(bpy)₂L(H₂O)]ⁿ⁺ and free NO. The second order rate constant for the second step of this reaction was calculated for cysteine k₂(SR⁻) = (2.20 ± 0.12) × 10⁹ M^− 1 s^− 1 and k_2(RSH) = (154 ± 2) M^− 1 s^− 1 for L = SO₃²⁻ and k₂(SR⁻) = (1.30 ± 0.23) × 10⁹ M^− 1 s^− 1 and k₂(RSH) = (0.84 ± 0.02) M^− 1 s^− 1 for L = imidazole; while for glutathione they were k₂(SR⁻) = (6.70 ± 0.32) × 10⁸ M^− 1 s^− 1 and k₂(RSH) = 11.8 ± 0.3 M^− 1 s^− 1 for L = SO₃²⁻ and k₂(SR⁻) = (2.50 ± 0.36) × 10⁸ M^− 1 s^− 1 and k₂(RSH) = 0.32 ± 0.01 M^− 1 s^− 1 for L = imidazole. In all reactions it was possible to detect the release of NO from the complexes, which it is remarkably distinct from other ruthenium metallocompounds described elsewhere with just N₂O production. These results shine light on the possible key role of NO release mediated by physiological thiols in reaction with these metallonitrosyl ruthenium complexes.     

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 × 10^− 9 cm²/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D? = 3.1 × 10^− 9 cm²/s) with a marked reduction after 30 min of TNF treatment (D? = 0.9 × 10^− 9 cm²/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.     

Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes

Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742-8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast k_on constants at 4.5 °C: 6.6 × 10⁸ M^− 1 s^− 1 for nNOS_726-749, 2.9 × 10⁸ M^− 1 s^− 1 for eNOS_492-511 and 6.1 × 10⁸ M^− 1 s^− 1 for iNOS_507-531, 3-4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca²⁺ chelation with ETDA: 3.7 s^− 1 for nNOS_726-749, 4.5 s^− 1 for eNOS_492-511, and 0.063 s^− 1 for iNOS_507-531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS_507-531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.     

Determination of the excitation migration time in Photosystem II: Consequences for the membrane organization and charge separation parameters

The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3 ± 1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes ~ 23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5 ± 0.4 ps)^− 1, the rate of secondary charge separation is (137 ± 5 ps)^− 1 and the drop in free energy upon primary charge separation is 826 ± 30 cm^− 1. These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].     

Polydopamine assisted fabrication of titanium oxide nanoparticles modified column for proteins separation by capillary electrochromatography

Development of a simple method for preparation of stable open tubular (OT) columns for proteins separation by capillary electrochromatography is still challenging. In this work, the titanium oxide (TiO₂) nanoparticles coated OT column was successfully prepared for separation of proteins by capillary electrochromatography. The polydopamine (PDA) film was first formed in the inner surface of a fused-silica capillary by the self-polymerization of dopamine under alkaline conditions. Then the TiO₂ coating was deposited onto the surface of pre-modified capillary with PDA by a liquid phase deposition process. The plentifully active hydroxyl groups in PDA coating can chelate with Ti⁴⁺ to boost the nucleation and growth of TiO₂ film. The as-prepared TiO₂ coated OT column was characterized by scanning electron microscopy and measurement of electroosmotic flow. Furthermore, the influence of liquid phase deposition time on the TiO₂ coating was investigated. The TiO₂ coated OT column was used for successful separation of two variants of β-lactoglobulin and eight glycoisoforms of ovalbumin. The column demonstrated good repeatability and stability. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.7%. Moreover, the application of the column was verified by successful separation of acidic proteins in egg white.     

Transferrin receptor-1 iron-acquisition pathway — Synthesis,kinetics, thermodynamics and rapid cellular internalization of a holotransferrin–maghemite nanoparticle construct

Background

Targeting nanoobjects via the iron-acquisition pathway is always reported slower than the transferrin/receptor endocytosis. Is there a remedy?

Methods

Maghemite superparamagnetic and theragnostic nanoparticles (diameter 8.6 nm) were synthesized, coated with 3-aminopropyltriethoxysilane (NP) and coupled to four holotransferrin (TFe₂) by amide bonds (TFe₂–NP). The constructs were characterized by X-ray diffraction, transmission electron microscopy, FTIR, X-ray Electron Spectroscopy, Inductively Coupled Plasma with Atomic Emission Spectrometry. The in-vitro protein/protein interaction of TFe₂–NP with transferrin receptor-1 (R1) and endocytosis in HeLa cells were investigated spectrophotometrically, by fast T-jump kinetics and confocal microscopy.

Results

In-vitro, R1 interacts with TFe₂–NP with an overall dissociation constant K_D = 11 nM. This interaction occurs in two steps: in the first, the C-lobe of the TFe₂–NP interacts with R1 in 50 μs: second-order rate constant, k₁ = 6 × 10¹⁰ M^− 1 s^− 1; first-order rate constant, k_− 1 = 9 × 10⁴ s^− 1; dissociation constant, K_1d = 1.5 μM. In the second step, the protein/protein adduct undergoes a slow (10,000 s) change in conformation to reach equilibrium. This mechanism is identical to that occurring with the free TFe₂. In HeLa cells, TFe₂–NP is internalized in the cytosol in less than 15 min.

Conclusion

This is the first time that a nanoparticle–transferrin construct is shown to interact with R1 and is internalized in time scales similar to those of the free holotransferrin.

General significance

TFe₂–NP behaves as free TFe₂ and constitutes a model for rapidly targeting theragnostic devices via the main iron-acquisition pathway.     

Mechanistic investigation of the oxidative addition reactions between different iridium (I) cyclooctadiene ([Ir(LL′)(cod)]) complexes and iodomethane

The oxidative addition reactions between two different [Ir(cod)(LL′)] complexes (LL′ = hpt and AnMetha) and iodomethane was kinetically investigated. The rate of oxidative addition was determined as 2.2(2) × 10⁻² and 2.69(6) × 10⁻² M⁻¹ s⁻¹ for [Ir(cod)(hpt)] and [Ir(cod)(AnMetha)] in nitromethane respectively. The large negative entropy of activation for the above-mentioned reactions in different solvents clearly point to an associative mechanism. An intrinsic volume of activation of −30.5(3) and −28(3) cm³ mol⁻¹ was determined for [Ir(cod)(hpt)] and [Ir(cod)(AnMetha)], respectively. A linear transition state with large charge separation and central ion contraction due to oxidation, contributes to the negative volume of activation.     

Analysis of proteins in microsamples of rat airway surface fluid by capillary electrophoresis

A thin layer of airway surface fluid (ASF) lining the pulmonary airways plays an important role in the primary defense mechanisms of the lung against bacterial infection. However, little is known about the composition of ASF due to the thinness (typically 5–30 μm in healthy animals) of the fluid layer and its relative inaccessibility, which causes considerable difficulties in sample collection and subsequent analysis. We have used a novel technique of capillary sampling coupled with capillary electrophoresis (CE) to analyze the protein composition of rat ASF. CE analyses were performed under two different conditions: a borate buffer, pH 9.1, or a phosphate buffer, pH 2.5, with 0.5 mM spermine. The different selectivities afforded by the two methods aid in peak identification, and quantitation of most of the major species was possible using both separation conditions. Albumin, transferrin and globulins are observed to be the major protein components in rat ASF, at concentrations of 28 mg ml⁻¹, 4.0 mg ml⁻¹ and 34 mg ml⁻¹ respectively, in comparison to 31 mg ml⁻¹, 3.1 mg ml⁻¹ and 40 mg ml⁻¹, respectively, in rat plasma.