Kinetic Studies Research Articles

Preparation and characterization of chitosan graphene oxide nanocomposite for the removal of 17-β-estradiol sulfate from water: kinetics, thermodynamics and simulation studies

The presence of 17-β-estradiol sulfate residue in water is of great concern due to its hazardous nature (endocrine disruptor and carcinogen). For this, chitosan was used with graphene oxide to prepare chitosan graphene oxide nanocomposite. This material was characterized and used as an adsorbent for the removal of 17-β-estradiol sulfate residue in water. This adsorbent showed a rapid, efficient, and environmentally friendly method of 17-β-estradiol sulfate removal from water. The optimal conditions for maximum removal were 93.6% (4.68 mg/g) with 2500 µg/L of 17-β-estradiol sulfate, 30 min of agitation, 0.50 g/L of graphene oxide chitosan nanocomposite, a pH of 7.0, and temperature of 25 °C. The thermodynamic parameters confirmed the spontaneity and exothermic nature of the 17-β-estradiol sulfate uptake process. The kinetic study revealed a first-order reaction, with the mechanism of uptake attributed to liquid film diffusion. The supra-molecular interaction between β-estradiol and graphene oxide chitosan nanocomposite was elucidated, highlighting the pivotal role of two hydrogen bonds with binding affinities of −6.80 and −7.03 kcal/mole for the first and second hydrogen bonds. This method is speedy, efficacy, and environmentally friendly; making it applicable for the removal of β-estradiol from water in diverse water systems.

Kinetic studies of AST isoenzymes I,II,III,IV partially purified from patient,s urine with chroinc renal failure

In this research, the kinetic studies of four isoenzymes of Asprtate aminotransferase, which partially purified from the urine of chronic renal failure patients were carried out .The four isoenzymes were obeyed Michaelis-Menton's equation and the optimum concentration of their substrate (Aspartic acid) was (166.5x10-3) mole/liter,and their Km values were determined. Four isoenzymesI,II,III,IV have shown an optimum pH at 7.4.The four isoenzymes obeyed Arrhenius equation up to 37º C and their Ea and Q10 constants were determined .

Crystal Structures of Arabidopsis thaliana Acetohydroxyacid Synthase in Complex with the Herbicide Triasulfuron and Two Analogues with Herbicidal Activity in Field Trials.

Triasulfuron is a commercial herbicide of the sulfonylurea family. This compound targets acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway. Here, we have determined crystal structures of Arabidopsis thaliana AHAS (AtAHAS) in complex with triasulfuron and two newly designed herbicidal compounds, identified as FMO and CMO, showing that their binding modes are subtly different. Kinetic studies showed all three compounds exhibit varying Ki values, 0.192 ± 0.013 μM for triasulfuron, 0.086 ± 0.013 μM for FMO, and 1.448 ± 0.058 μM for CMO, but all are strong time-dependent accumulative inhibitors of AtAHAS. Apart from triasulfuron being a powerful herbicide with application rates of 10-15 g/ha in wheat fields, CMO and FMO are also herbicidal at 7.5-30 g/ha for barnyard grass. This study emphasizes that accumulative inhibition is an important factor that contributes to herbicidal activity.

Adsorption Study for Chromium (VI) on Iraqi Bentonite

The subject of this research involves studying adsorption to remove hexavalent chromium Cr(VI) from aqueous solutions. Adsorption process on bentonite clay as adsorbent was used in the Cr(VI) concentration range (10-100) ppm at different temperatures (298, 303, 308 and 313)K, for different periods of time. The adsorption isotherms were obtained by obeying Langmuir and Freundlich adsorption isotherm with R2 (0.9921-0.9060) and (0.994-0.9998), respectively. The thermodynamic parameters were calculated by using the adsorption process at four different temperatures the values of ?H, ?G and ?S was [(+6.582 ? +6.547) kJ.mol-1, (-284.560 ? -343.070) kJ.mol-1 and (+0.977 ? +1.117) kJ.K-1.mol-1] respectively. This data indicates the spontaneous sorption process. The kinetic study of adsorption process was studied depending on three kinetic equations: 1- Lagergren equation 2- Morris-Weber equation 3- Reichenberg equation

Intrinsic Metal Component-Assisted Microwave Pyrolysis and Kinetic Study of Waste Printed Circuit Boards

Waste printed circuit boards (WPCBs) hold great recycling value, but improper recycling can lead to environmental issues. This study combines pyrolysis and microwave technologies, leveraging the unique phenomenon where metal materials tend to “spark” in a microwave field, to develop a microwave pyrolysis process for WPCBs that incorporates metal fillers. The research analyzes the effects of microwave power, metal filler addition, and pyrolysis time on the efficiency of microwave pyrolysis. It explores the mechanisms of microwave pyrolysis and the pathways of pyrolysis product formation, and the kinetics of the pyrolysis reaction of WPCBs. The results indicate that microwave-assisted pyrolysis greatly improves efficiency. Within the experimental range, the optimal conditions are found to be a microwave power of 1600–1800 W, a metal filler addition of 10%, and a pyrolysis time of 10 min. Under these conditions, the yield of pyrolysis liquid was 12.8%, with approximately 5–12 different components, while the yield of pyrolysis gas was 12.7–13.4%, with about 9–11 different components. Compared to conventional pyrolysis products, the liquid products from microwave pyrolysis are simpler and more advantageous for resource utilization. Theoretical calculations show that the average activation energy for the microwave pyrolysis process is 81.05 kJ/mol, with an average reaction order of 0.93, which is greatly better than the 147.75 kJ/mol of the conventional pyrolysis process.

The combined effects of polystyrene nanoplastics and dissolved organic matter on the environmental bioavailability of carbamazepine

The bioavailability of active pharmaceutical ingredients (APIs) plays a crucial role in determining the toxicity and risk of contaminants in the environment. However, the bioavailability of APIs in complex environmental matrices is still unclear. In this study, the combined effects of polystyrene nanoplastics (PS NPs) with various particle sizes (50, 100, and 1000 nm) and fulvic acid (FA) on the bioavailability of carbamazepine (CBZ) were investigated via negligible depletion solid-phase microextraction (nd-SPME) and Daphnia magna (D. magna) accumulation. The uptake kinetic study revealed that both PS NPs and FA reduced the elimination rate (k2) in most cases. The availability of CBZ to nd-SPME was determined by the hydrodynamic particle size of PS NPs, whereas the bioavailability to D. magna depended on the intrinsic particle size. The CBZ bioavailability was greater in co-exposed matrices due to the attenuated sorption of PS NPs to CBZ by FA modification. Notably, co-exposure of PS NPs and FA resulted in a higher bioaccumulation factor (BAF) of CBZ, probably due to the desorption and reabsorption of particle-associated CBZ. This study demonstrated that both PS NP particle size and FA binding affect the bioavailability of CBZ, and nd-SPME can mimic only the bioaccumulation of CBZ via diffusion.

A facile and green synthesis of corn cob-based graphene oxide and its modification with corn cob-K2CO3 for efficient removal of methylene blue dye: Adsorption mechanism, isotherm, and kinetic studies

In recent years, the treatment of synthetic dyes has become an environmental concern. In this study, a single step calcination process was used to develop the inventive, simple, and inexpensive adsorbent CC-GO/CC-K2CO3 composite. The composite was employed for the treatment of methylene blue (MB), a cationic dye. Several characterization methods including powder XRD, FTIR, XPS, BET, FESEM, EDX, Raman, and HRTEM techniques were utilized for the analysis of the composite. The surface area and mean pore diameter of CC-GO/CC-K2CO3 were 32.651 m2 g−1 and 3.71 nm, respectively. The adsorption experiment showed that optimal parameters for the removal of MB dye are at an adsorbent dose of 60 mg, initial dye concentration of 80 mg/L, contact time of 150 min, and pH value of 12 at room temperature. Under optimized conditions, CC-GO evidences a removal efficiency of 70.34 ± 1.36 % while after incorporation with CC-K2CO3 the removal capacity sharply increases up to 98.10 ± 0.4 %. Kinetic and isotherm models were used to analyze the removal rate constant and equilibrium adsorption capacity under various adsorption environments. The adsorption study was found to follow the models of pseudo-second order kinetic and Freundlich isotherm. The CC-GO/CC-K2CO3 composite has the maximum adsorption capacity (MAC) of 160.77 mg/g established by the Langmuir isotherm. The prepared composite has demonstrated the capacity to be recycled up to three times with a gradual decrease in its adsorption behavior, exhibiting removal efficiency of 61.66 ± 2.04 %.A cost estimation study of the composite was also performed to assess its cost effectiveness.

Kinetic and isotherm adsorption studies of Zn (II) ions on hydroxyapatite nanoparticles: Linear and nonlinear analyses

Kinetic and isotherm adsorption studies of Zn (II) ions on hydroxyapatite nanoparticles: Linear and nonlinear analyses

Design, synthesis and enzymatic inhibition evaluation of novel 4-hydroxy Pd–C-Ⅲ derivatives as α-glucosidase and PTP1B dual-target inhibitors

A library of 4-Hydroxy Pd–C-Ⅲ derivatives (5a-5p and 8a-8h) as α-glucosidase inhibitors was prepared and the activity of these compounds against α-glucosidase was evaluated. The outcomes displayed that most of the derivatives had moderate to potent α-glucosidase inhibition with IC50 values ranging from 66.3 ± 2.4 to 299.7 ± 6.0 μM. Amongst these compounds, 8a had the strongest α-glucosidase inhibition than others with an IC50 value of 66.3 ± 2.4 μM. Therefore, 8a was chosen to detect the inhibitory activities on PTP1B and α-amylase, the results revealed that 8a had the potential to be PTP1B (IC50 = 47.0 ± 0.5 μM) and α-amylase (IC50 = 30.62 ± 2.13 μM) inhibitor. Additionally, the enzyme kinetic study displayed that 8a was a mixed-type inhibitor. Moreover, the results of the spectroscopy experiments proved that 8a could quench the fluorescence intensity of α-glucosidase in a dose-dependent manner, destroy the secondary structure of α-glucosidase and change the conformation of the enzyme. Significantly, the investigation of cellular thermal shift assay exhibited that 8a could target the PTP1B protein, and the in vitro cytotoxicity discovered compound 8a had no significant toxicity to normal HEK-293 cells. Additionally, the results of molecular docking found that 8a could both bind the active sites of the α-glucosidase and PTP1B. Importantly, the in vivo sucrose-loading test displayed 8a had potential to reduce the postprandial blood glucose. All results proved that compound 8a had great potential as a dual-target inhibitor in treating Type 2 diabetes mellitus.

Enhancing the biotransformation of progesterone to the anticancer compound testololactone by Penicillium chrysogenum Ras3009: kinetic modelling and efficiency maximization

BackgroundBiotransformation of steroid compounds into therapeutic products using microorganisms offers an eco-friendly and economically sustainable approach to the pharmaceutical industry rather than a chemical synthesis way. The biotransformation efficiency of progesterone into the anticancer compound testololactone using Penicillium chrysogenum Ras3009 has been investigated. Besides, maximization of testololactone formation was achieved by studying the kinetic modelling and impact of some fermentation conditions on the biotransformation process.ResultsThe fungal strain Ras3009 was selected among twelve fungal strains as the most runner for the transformation of 81.18% of progesterone into testololactone. Ras3009 was identified phenotypically and genotypically as Penicillium chrysogenum, its 18 S rRNA nucleotide sequence was deposited in the GenBank database by the accession number OR480104. Studying the impact of fermentation conditions on biotransformation efficiency indicated a positive correlation between substrate concentration and testololactone formation until reaching the maximum velocity vmax. Kinetic studies revealed that vmax was \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\:0.0482$$\\end{document} gL− 1hr− 1 with high accuracy, giving R2 of 0.977. The progesterone transformation efficiency generally increased with time, reaching a maximum of 100% at 42 h with testololactone yield (Ypt/s) 0.8700 mg/mg. Moreover, the study indicated that the enzymatic conversion by P. chrysogenum Ras3009 showed high affinity to the substrate, intracellularly expressed, and released during cell disruption, leading to higher efficiency when using whole microbial cell extract.ConclusionsFungi can be promising biocatalysts for steroid transformation into valuable chemicals and pharmaceutical compounds. The study revealed that the new fungal isolate P. chrysogenum Ras3009 possesses a great catalytic ability to convert progesterone into testololactone. Kinetic modelling analysis and optimization of the fermentation conditions lead to higher transformation efficiency and provide a better understanding of the transformation processes.Graphical abstract

Preparation of new green poly (amino amide) based on cellulose nanoparticles for adsorption of Congo red and its adaptive neuro-fuzzy modeling

In this study, a novel green poly(amino amide) nanoparticle based on cellulose nanoparticles (Cell-PAMN) was developed for the efficient adsorption of Congo Red dye. Cellulose nanocrystals obtained from acid hydrolysis of cotton linter were functionalized via Oxa-Michael addition of acrylamide on their surface hydroxyl groups, followed by transamidation with ethylenediamine. The resulting nanoparticles were characterized using FT-IR spectroscopy, SEM, and X-ray diffraction techniques. The as-prepared Cell-PAMN exhibited considerably higher adsorption capacity compared to unmodified cellulose nanoparticles due to the presence of amino and amide functional groups. The adsorption kinetics and the effects of parameters such as contact time and initial dye concentration on the adsorption capacity were investigated. An adaptive Neuro-Fuzzy model was used to study the efficiency of dye removal, accurately predicted the adsorption behavior of Cell-PAMN. The kinetic study results showed that the adsorption process followed a pseudo-second-order kinetic model, with a maximum adsorption capacity of around 40 mg/g. The results demonstrated the potential of the synthesized material for the removal of Congo Red from aqueous solutions, highlighting its applicability in wastewater treatment. This research contributes to the development of sustainable and eco-friendly materials for environmental remediation applications.

Efficient elimination of 3-monochloropropane-1,2-diol fatty acid ester from palm oil via using activated carbon grafted with Tween80.

In the current study, we attempt to remove 3-monochloropropane-1,2-diol fatty acid ester (3-MCPD ester) from palm oil with developed composite adsorbent (Tween80 modified activated carbon [AC]), and scanning electron microscopy-energy dispersive spectrometer, Fourier transform-infrared spectroscopy, X-ray diffraction, nitrogen content adsorption-desorption and thermogravimetric analysis were used to characterize the modifications. We further examined the adsorption capability of the composite adsorbent for 3-MCPD ester and found that the highest removal efficiency was 87.36% (5.3% of adsorbent dose at 104°C for 29min). This is approximately three times higher than that of pristine AC, implying that the composite can be employed as a novel adsorbent for 3-MCPD ester reduction. Along with the adsorption mechanism of Langmuir, Freundlich, Dubinin-Radushkevich, and Temkin models were also tested. It has been suggested that Freundlich model could best describe the adsorption process. Adsorption was found to be well-fitted by pseudo-second-order kinetics according to the kinetic study. An endothermic and spontaneous adsorption mechanism was demonstrated by the thermodynamic studies.

Structural insights into alterations in the substrate spectrum of serine-β-lactamase OXA-10 from Pseudomonas aeruginosa by single amino acid substitutions

ABSTRACT The extensive use of β-lactam antibiotics has led to significant resistance, primarily due to hydrolysis by β-lactamases. OXA class D β-lactamases can hydrolyze a wide range of β-lactam antibiotics, rendering many treatments ineffective. We investigated the effects of single amino acid substitutions in OXA-10 on its substrate spectrum. Broad-spectrum variants with point mutations were searched and biochemically verified. Three key residues, G157D, A124T, and N73S, were confirmed in the variants, and their crystal structures were determined. Based on an enzyme kinetics study, the hydrolytic activity against broad-spectrum cephalosporins, particularly ceftazidime, was significantly enhanced by the G157D mutation in loop 2. The A124T or N73S mutation close to loop 2 also resulted in higher ceftazidime activity. All structures of variants with point mutations in loop 2 or nearby exhibited increased loop 2 flexibility, which facilitated the binding of ceftazidime. These results highlight the effect of a single amino acid substitution in OXA-10 on broad-spectrum drug resistance. Structure–activity relationship studies will help us understand the drug resistance spectrum of β-lactamases, enhance the effectiveness of existing β-lactam antibiotics, and develop new drugs.

Synthesis of a New Composite Material Derived from Cherry Stones and Sodium Alginate—Application to the Adsorption of Methylene Blue from Aqueous Solution: Process Parameter Optimization, Kinetic Study, Equilibrium Isotherms, and Reusability

Synthesis of a New Composite Material Derived from Cherry Stones and Sodium Alginate—Application to the Adsorption of Methylene Blue from Aqueous Solution: Process Parameter Optimization, Kinetic Study, Equilibrium Isotherms, and Reusability

Investigating the inhibition of xanthine oxidase by five catechins: Kinetic studies, spectroscopy, molecular docking, and dynamics simulations

Catechins compounds from tea have demonstrated significant inhibitory effects on xanthine oxidase (XOD). However, the precise inhibitory mechanisms of the main catechins on XOD remain to be fully elucidated. This study explored the inhibition mechanisms and binding characteristics of five catechins (GC, EGC, EC, EGCG, and ECG) on XOD through a combination of inhibition kinetics, multi-spectroscopy analysis, molecular docking, and dynamics simulations. Among the catechins, EGCG and ECG exhibited the most potent inhibitory activities against XOD. All five catechins were found to exhibit mixed inhibition, affecting the hydrophobic groups and secondary structure of XOD predominantly through hydrophobic interactions and hydrogen bonding. Molecular dynamics simulations revealed that a 3,4,5-trihydroxybenzoic acid moiety at C3 position significantly enhances the binding affinity of EGCG and ECG to XOD. Additionally, the decrease of β-sheet and random coil induced by EGCG and ECG was found to be crucial for enhancing inhibitory activity of XOD. In vitro cell experiments showed that EGCG and ECG significantly reduced high uric acid levels of BRL-3A cell. This study elucidates the inhibitory mechanisms of catechins on XOD, paving the way for their application as XOD inhibitors to combat hyperuricemia.

Quercetin as a Modulator of PTPN22 Phosphomonoesterase Activity: A Biochemical and Computational Evaluation.

Cancer, a group of diseases characterized by uncontrollable cell proliferation and metastasis, remains a global health challenge. This study investigates quercetin, a natural compound found in many fruits and vegetables, for its potential to inhibit the phosphomonoesterase activity of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), a key immune response regulator implicated in cancer and autoimmune diseases. We started by screening seven (7) natural compounds against the activities of PTPN22 in vitro. The initial screening identified quercetin with the highest percentage inhibition (81%) among the screened compounds when compared with ursolic acid that has 84%. After the identification of quercetin, we proceeded by investigating the effect of increasing concentrations of the compound on the activity of PTPN22. In vitro studies showed that quercetin inhibited PTPN22 with an IC50 of 29.59 μM, outperforming the reference standard ursolic acid, which had an IC50 of 37.19 μM. Kinetic studies indicated a non-competitive inhibition by quercetin with a Ki of 550 μM. In silico analysis supported these findings, showing quercetin's better binding affinity (ΔGbind -24.56 kcal/mol) compared to ursolic acid, attributed to its higher reactivity and electron interaction capabilities at PTPN22's binding pocket. Both quercetin and ursolic acid improved the structural stability of PTPN22 during simulations. These results suggest quercetin's potential as an anticancer agent, meriting further research. However, in vivo studies and clinical trials are necessary to fully assess its efficacy and safety, and to better understand its mechanisms of action.

Effect of 4,4′-dialkyl-2,2′-bipyridine ligands on the hydrolysis of dichlorodioxomolybdenum(VI) catalyst precursors and the switch from homogeneous epoxidation to heterogeneous systems

Molybdenum catalysts have been industrially recognized for decades for liquid phase epoxidation, which is an important chemical reaction process, since epoxides are used for many industrial applications. In this work, molybdenum oxide hybrid catalysts, prepared by a reflux hydrolysis methodology, performed effectively as heterogeneous catalysts or reaction-induced self-separating catalysts under mild reaction conditions; in the two cases, the catalyst separation and reuse are facilitated. Specifically, catalysts with the general formula [MoO3(L)], possessing polymeric chain-like (L = 4,4′-dimethyl-2,2′-bipyridine (1)) or oligomeric (L = 4,4′-dinonyl-2,2′-bipyridine (2)) structures comprising corner-sharing {MoO4N2} units, were synthesized and characterized by various complementary techniques (ATR FT-IR, Raman, 13C{1H} CP MAS NMR spectroscopy, PXRD, SEM, TGA, elemental analysis, ICP-OES and N2 sorption isotherms). Small chemical differences in the organic synthesis precursor had important structure directing effects on the type of hybrid material formed. The hybrids promoted olefin epoxidation with H2O2 or tert-butylhydroperoxide (TBHP) as oxidant. For example, 1 catalyzed the conversion of biobased olefins (70 °C) and lignin-based isoeugenol (50 °C) with TBHP to useful bioproducts, in heterogeneous phase, leading to an epoxide yield of 100 % for DL-limonene (3:1 M molar ratio of 1,2-epoxy-p-menth-8-ene to 1,2:8,9-diepoxy-p-menthane), 81 % epoxide yield for fatty acid methyl esters, and 80 % Licarin A selectivity at 40 % isoeugenol conversion. For dienes (DL-limonene, methyl linoleate), kinetic modelling studies suggested that the formation of the monoepoxides was faster than that of diepoxides, accounting for enhanced monoepoxide selectivity.

Visible Light-Triggered Catalytic Performance of Reduced Graphene Oxide Decorated With Copper Oxide Nanocomposite for Degradation of Rhodamine B Dye and Kinetics Studies.

Herein, novel nanocomposites based on reduced graphene oxide decorated copper oxide nanoparticles (rGO/CuO) were prepared by the in situ co-precipitation method. The structural, morphological, and optical characterization of as-prepared nanocomposites was performed by powdered x-ray diffraction (p-XRD), scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR), Raman, and ultraviolet-visible (UV-Vis) spectroscopy, respectively. The as-prepared nanocomposites exhibited better photocatalytic activity of rhodamine B dye with maximum ~94% degradation in 120 min with a rate constant of 0.2353 min-1 under optimized conditions. Furthermore, the effects of solution pH and catalyst loading are studied on the degradation process. Therefore, this state-of-the-art strategy for the decoration of CuO nanoparticles onto the surface of rGO nanosheets could be an ideal platform for fabricating highly efficient photocatalysts.

Synthesis of hydrotalcite and titanium dioxide (HT-TiO2) composite for application in heterogeneous photocatalytics processes

The objective of the study was to synthesize a composite of hydrotalcite and titanium dioxide (HT-TiO2) for application in heterogeneous photocatalytic processes to the degradation of organic pollutants in aqueous media. The composites were characterized by X-ray diffraction and surface area, then an experimental design was carried out to evaluate the influence of pH and temperature on the adsorption capacity of pollutants against the anionic dye indigo carmine using a kinetic study. Photocatalytic tests were carried out in a chamber equipped with a UV lamp and hydrogen peroxide as an auxiliary oxidant in degradation. The adsorption results corroborated with the order of surface areas obtained, where the adsorption capacity followed the order of HT, HT-TiO2 5 %, HT-TiO2 10 %, and TiO2, respectively, obtaining removals up to 96.94 %. For the heterogeneous photocatalytic tests, the best conditions of the experimental design were used for each semiconductor, with a removal of up to 99.96 % being observed in 2 h. The use of hydrogen peroxide as an auxiliary oxidant reduced the reaction time to 45 min, demonstrating how joint effluent treatment techniques can increase the speed and efficiency of the process.

Rationalizing iron-gall ink recipes from antiquity until the 19th century. A kinetic study.

Iron-gall inks, a vital part of our written cultural heritage, are at risk of complete loss due to degradation, a potential loss that we must urgently address. These inks are based on Fe3+-complexes with phenolic compounds, which grow to form a complex network of iron oxyhydroxides. Over time, these black inks turn into brownish tones, with extensive degradation in paper support leading to extensive breaking. The kinetics of iron-gall ink preparation explains the use of iron sulfate, FeSO4, in all ancient recipes to obtain a stable amorphous ink. The novelty of this work shows that a low ratio of Fe3+/polyphenol is a crucial factor in allowing the ink's growth without its degradation. This ratio also prevents the formation of superoxide. This was achieved through a comprehensive research methodology involving spectroscopic techniques in the visible and the near-infrared regions, stopped-flow spectrometry and electrochemical studies.