Kinetics Of Enzymatic Reaction Research Articles

How does methylviologen cation radical supply two electrons to the formate dehydrogenase in the catalytic reduction process of CO2 to formate?

Formate dehydrogenase from Candida boidinii (EC.1.2.1.2; CbFDH) is a commercially available enzyme and can be easily handled as a catalyst for the CO2 reduction to formate in the presence of NADH, single-electron reduced methylviologen (MV+˙) and so on. It was found that the formate oxidation to CO2 with CbFDH was suppressed using the oxidized MV as a co-enzyme and the single-electron reduced MV (MV+˙) was effective for the catalytic activity of CbFDH for the CO2 reduction to formate compared with that using the natural co-enzyme of NADH [Y. Amao, Chem. Lett., 2017, 46, 780-788]. The CO2 reduction to formate catalyzed by CbFDH requires two molecules of the MV+˙. In order to clarify the two-electron reduction process using MV+˙ in the CO2 reduction to formate catalyzed with CbFDH, we attempted enzyme reaction kinetics, electrochemical and quantum chemical analyses. Kinetic parameters obtained from the enzymatic kinetic analysis metric revealed an index of affinity of MV+˙ for CbFDH in the CO2 reduction to formate. From the results of the electrochemical analysis, it was predicted that only one molecule of MV+˙ was bound to CbFDH, and the MV bound to CbFDH was to be necessarily re-reduced by the electron source outside of CbFDH to supply the second electron in the CO2 reduction to formate. From the results of docking simulation and density functional theory (DFT) calculations, it was indicated that one molecule of MV bound to the position close to CO2 in the inner part of the substrate binding pocket of CbFDH contributed to the two-electron CO2 reduction to formate.

Phenoxazinone synthase mimicking activity of a dinuclear copper(II) complex with a half salen type Schiff base ligand

A dinuclear copper(II) complex [LCu(μ1,1-N3)2CuL] {where HL = (2-(3-aminopropylimino)methyl-6-methoxyphenol)} has been synthesized and characterized by several analytical techniques including single crystal X-ray diffraction analysis. Interesting supramolecular interactions are observed in the solid state of the complex. An unusual N–H⋯π interaction is observed where the chelate ring, Cu(1)–N(1)–Cu(1)b–N(1)b, [where b = 1 − x, −y, 1 − z] behaves as a π system. Supramolecular interactions have been related with Hirshfeld surface calculations. The complex has been evaluated as functional model for phenoxazinone synthase enzyme by using o-aminophenol (OAPH) as a model substrate in acetonitrile medium. Kinetics of the reaction was followed spectrophotometrically, which confirm that catalytic reactions follow Michaelis–Menten enzymatic reaction kinetics.

Efficacy of spectroscopic optical coherence tomography (sOCT) for instantaneous study of nitrilase enzyme kinetics

We present monitoring of nitrilase enzyme assay in real-time by means of Spectroscopic Optical Coherence Tomography (sOCT). Combination of principal component analysis (PCA) algorithm with Swept Source Optical Coherence Tomography (SSOCT), to extract the absorption spectra of the sample, termed as sOCT. Spectroscopic information of nitrilase enzyme obtained from sOCT is used to investigate dynamic enzyme kinetic reactions. Different principle components give complete information regarding the composition of the samples. Further, Fourier Transform Infrared Spectroscopy (FTIR) analysis has been also performed to validate the PCA absorption spectra. Samples were prepared in controlled condition and evaluated at a regular time interval of the incubation period. The developed algorithm and system are combinedly capable of in-vitro real-time, study of enzyme kinetics with respect to time as biosensor.

The synthesis, characterization, DNA/protein interaction, molecular docking and catecholase activity of two Co(II) complexes constructed from the aroylhydrazone ligand

Abstract Two Co(II) complexes, Co(BBH)phenMeCN (1) and Co(BSH)phenMeCN (2) (HBBH = benzophenone benzoyl hydrazone, HBSH = benzophenone salicylylhydrazone ligand and phen = 1,10-phenanthroline) were prepared and characterized by single X-ray crystallography. The investigation of the complexes binding with herring sperm DNA (HS-DNA) revealed that stronger binding interaction exists between the complexes and HS-DNA compared to the corresponding free ligands. And both complexes can bind to DNA through intercalation of the phenyl rings between adjacent base pairs in the double helix. Meanwhile, bovine serum albumin (BSA) binding studies reveal the complexes can effectively interact with BSA and change the secondary structure of BSA. Superoxide dismutase (SOD) mimic activity of the complexes was investigated and the results showed that complex 2 showed a stronger radical scavenging potency against O2.- radical with an IC50 value of 16.10 μM. The catecholase-like activity of both complexes has been investigated with 3,5-di-tert-butylcatechol (3,5-DTBC) as the substrate in methanol solution under completely aerobic conditions. The catalytic reaction follows Michaelis-Menten enzymatic reaction kinetics with turnover numbers (kcat) of 34.14 h−1 and 30.36 h−1 for complex 1 and complex 2, respectively.

Static microdroplet array generated by spraying and analyzed with automated microscopy and image processing

Microdroplets have received increasing interest as practical platforms for high-throughput biochemical analysis. Typically, numerous discrete aqueous microdroplets containing biochemical targets are generated in a continuous oil phase and characterized using a flow-through configuration. Although this approach is capable of extremely high throughput, it is challenging to provide dynamic characterization of time-dependent reaction kinetics. In this paper, we present a practical and affordable method to create and analyze a massive array of static aqueous microdroplets immersed in oil for biochemical analysis. The discrete microdroplets were produced by an air-spray gun, imaged by automated microscopy, and then characterized by image processing. The location, area, and fluorescence intensity of randomly generated individual microdroplets were automatically registered for high-throughput characterization. With this approach, we rapidly produced and characterized a static microdroplet array of over 0.7 million microdroplets with an average volume of 300 fL and a mean population density of 1.5*105 microdroplets/cm2. Using the developed setup, we demonstrated the dependency of the microdroplets’ fluorescence intensity on their volume, as well as characterized the time-dependent enzyme reaction kinetics of β-galactosidase-mediated cleavage of the substrate fluorescein di-β-d-galactopyranoside (FDG). The new approach described herein provides an inexpensive alternative solution for high-throughput analysis of dynamic biochemical processes.

In situ oxidation of canola meal sinapic acid by horseradish peroxidase (type II) and tyrosinase.

The enzymatic oxidation of sinapic acid catalyzed by horseradish peroxidase (HRP) or tyrosinase was investigated using model systems, which contained the pure compound or canola meal. Spectrophotometric scanning of pure sinapic acid solution in the presence of HRP (0.2 U) or tyrosinase (40.3 U) showed continuous decreases in absorbance at 304nm over a period of 90 and 60min, respectively. HPLC analyses of enzymatic end products, obtained by the catalysis with HRP or tyrosinase, indicated the presence of two main compounds (1 and 2). After alkaline hydrolysis of canola meal, sinapic acid that was released from sinapine was also converted to compounds 1 and 2 by HRP or tyrosinase. Enzyme reaction kinetics results indicate that the catalytic efficiency (CE=0.538), reaction velocity (Vmax =5.67 ∆A/h), and Michaelis-Menten constant (Km =926.64µM) of HRP are significantly higher than those of tyrosinase (CE=0.041, Vmax =0.41 ∆A/h, Km =173.03µM) at 50-250μM pure sinapic acid concentrations. PRACTICAL APPLICATIONS: Canola meal contains a large amount of sinapine, which is the choline ester of sinapic acid, a strong antioxidant compound. However, the oxidation or decarboxylation products of sinapic acid could add value by increasing the level of electron-dense carboxylic and carbonyl compounds. In this study, enzymatic treatment of alkaline-hydrolyzed canola meal with horseradish peroxidase (HRP) and tyrosinase was investigated and shown to be suitable for converting sinapic acid into oxidized compounds. Therefore, the enzymatic treatment is a potential application for value-added processing of canola meal.

A bombykol electrochemical receptor sensor and its kinetics

This study aimed to explore the interaction between bombykol and BmOR1 and also provide a paradigm for agroforestry pest control. The electrochemical biosensor signal amplification system was used: nanogold with horseradish peroxidase. An electrochemical bilayer nanogold membrane receptor sensor was developed using the following schemes and processes: twice self-assembly of nanogold and succeeding absorption of Bombyx mori olfactory receptor 1 (BmOR1); sex pheromone–binding protein; spectral scanning and transmission electron microscope to characterize nanogold sol; and atomic force microscope, cyclic voltammetry, and AC impedance methods to characterize individual processes of sensor assembly. The amperometric I–T curve was adopted to measure the response current upon interaction with different concentrations of bombykol (diluted in phosphate-buffered saline) and BmOR1. The results demonstrated the receptor–ligand interaction pattern, which was similar to enzymatic reaction kinetics, with the activation constant Ka of up to 8.57 × 10−20 mol/L and signal magnification of about 10,000-fold. In this study, the simulation of intracellular receptor signaling cascade by an electrochemical signal amplification system helped in directly measuring BmOR1–bombykol ligand interaction and exploring the kinetics after the self-assembly of BmOR1 on the biosensor. It provided a novel platform for future studies on receptor–ligand interaction.

General mathematical formula for near equilibrium relaxation kinetics of basic enzyme reactions and its applications to find conformational selection steps

A general mathematical formula of basic enzyme reactions was derived with nearly no dependence on conditions nor assumptions on relaxation kinetic processes near equilibrium in a simple single-substrate-single-product enzyme reaction. The new formula gives precise relationships between the rate constants of the elementary reaction steps and the apparent relaxation rate constant, rather than the initial velocity that is generally used to determine enzymatic parameters according to the Michaelis–Menten theory. The present formula is shown to be complementary to the Michaelis–Menten formulae in a sense that the initial velocity and the relaxation rate constant data together could determine the enzyme–substrate dissociation constant KES, which has been usually conditionally approximated by the Michaelis constant KM within the framework of the Michaelis–Menten formulae. We also describe relaxation kinetics of enzyme reactions that include the conformational selection processes, in which only one enzymatic conformer among a conformational ensemble can bind with either the substrate or product. The present mathematical approaches, together with numerical computation analyses, suggested that the presence of conformational selection steps in enzymatic reactions can be experimentally detected simply by enzymatic assays with catalytic amounts of enzyme.

A novel wide-range microfluidic dilution device for drug screening.

Microfluidic dilution chip is a crucial approach to perform gradient dilution of experimental samples in many biological investigations. In this study, we developed two serial wide-range dilution chips with dilution rates of 1:1 and 1:4 on the basis of the microfluidic oscillator by designing a series chamber, which was similar to a series circuit. The size of this chamber was adjusted and mixed with the neighboring air chamber to form dilution rates by oscillatory methods. We applied this microfluidic oscillator to estimate cellular kinetics and perform an acute oxidative stress test on Caenorhabditis elegans (C. elegans) in order to further validate their effectiveness. We estimated the kinetic parameters of β-galactosidase, the biocatalyst responsible for the hydrolysis of lactose, and found out that K m was 602 ± 73 μM and k cat was 72 ± 12/s. In addition, our result of the study on acute oxidative stress of C. elegans using this novel chip was consistent with the result using 96-well plates. Overall, we believe that this novel chip can be applied to enzymatic reaction kinetics to evaluate accurately drug screening in bio-nematode models such as C. elegans. In summary, we have provided a novel microfluidic dilution chip that can form a wide range of sample concentration gradients. Our chip may facilitate drug screening, drug toxicology, and environmental toxicology.

New Aspects of Protein‐film Voltammetry of Redox Enzymes Coupled to Follow‐up Reversible Chemical Reaction in Square‐wave Voltammetry

AbstractProtein‐film square‐wave voltammetry of uniformly adsorbed molecules of redox lipophilic enzymes is applied to study their electrochemical properties, when a reversible follow‐up chemical reaction is coupled to the electrochemically generated product of enzyme's electrode reaction. Theoretical consideration of this so‐called “surface ECrev mechanism” under conditions of square‐wave voltammetry has revealed several new aspects, especially by enzymatic electrode reactions featuring fast electron transfer. We show that the rate of chemical removal/resupply of electrochemically generated Red(ads) enzymatic species, shows quite specific features to all current components of calculated square‐wave voltammograms and affects the electrode kinetics. The effects observed are specific for this particular redox mechanism (surface ECrev mechanism), and they got more pronounced at high electrode kinetics of enzymatic reaction. The features of phenomena of “split net‐SWV peak” and “quasireversible maximum”, which are typical for surface redox reactions studied in square‐wave voltammetry, are strongly affected by kinetics and thermodynamics of follow‐up chemical reaction. While we present plenty of relevant voltammetric situations useful for recognizing this particular mechanism in square‐wave voltammetry, we also propose a new approach to get access to kinetics and thermodynamics of follow‐up chemical reaction. Most of the results in this work throw new insight into the features of protein‐film systems that are coupled with chemical reactions.

Kinetic modelling and techno-economic analysis of biodiesel production from Calophyllum inophyllum oil

Calophyllum inophyllum oil, a non-edible renewable resource, was used for the production of biodiesel. In the present study, kinetic analysis was done for the biodiesel production from C. inophyllum oil using alkali and enzymatic method, a non-edible renewable resource with methanol to identify the rate equation and estimate model kinetic parameters. The present model was validated, and the experimental and predicted values were compared. The first-order rate equation represents an alkali transesterification reaction’s kinetic mathematical model under chosen experimental conditions for the estimated parameters. The enzymatic transesterification was carried out at 500 rpm for 8 h in room temperature produced a yield of 97% of FAME (fatty acid methyl ester) for the reaction with 6:1 methanol to oil in molar ratio. The sequential cleavage of fatty acid and reaction with methanol by the enzyme were assumptions used in developing the kinetic model. The transesterification of C. inophyllum oil was simulated with Aspen Plus (V 8.6, Aspen Tech., Inc.). The simulation and economically viable estimation for the comparison of a ceaseless biodiesel production from C. inophyllum oil are formed on the basis of the kinetics of chemical transesterification (base-catalyzed methanolysis) and enzymatic transesterification reactions. The base-catalyzed transesterification requires nearly seven times higher than the enzymatic transesterification Also, the simulation demonstrated that the base-catalyzed transesterification required additional number of process equipment units when related to the enzymatic process.

Computational characterization of enzyme-bound thiamin diphosphate reveals a surprisingly stable tricyclic state: implications for catalysis.

Thiamin diphosphate (ThDP)-dependent enzymes constitute a large class of enzymes that catalyze a diverse range of reactions. Many are involved in stereospecific carbon–carbon bond formation and, consequently, have found increasing interest and utility as chiral catalysts in various biocatalytic applications. All ThDP-catalyzed reactions require the reaction of the ThDP ylide (the activated state of the cofactor) with the substrate. Given that the cofactor can adopt up to seven states on an enzyme, identifying the factors affecting the stability of the pre-reactant states is important for the overall understanding of the kinetics and mechanism of the individual reactions.In this paper we use density functional theory calculations to systematically study the different cofactor states in terms of energies and geometries. Benzoylformate decarboxylase (BFDC), which is a well characterized chiral catalyst, serves as the prototypical ThDP-dependent enzyme. A model of the active site was constructed on the basis of available crystal structures, and the cofactor states were characterized in the presence of three different ligands (crystallographic water, benzoylformate as substrate, and (R)-mandelate as inhibitor). Overall, the calculations reveal that the relative stabilities of the cofactor states are greatly affected by the presence and identity of the bound ligands. A surprising finding is that benzoylformate binding, while favoring ylide formation, provided even greater stabilization to a catalytically inactive tricyclic state. Conversely, the inhibitor binding greatly destabilized the ylide formation. Together, these observations have significant implications for the reaction kinetics of the ThDP-dependent enzymes, and, potentially, for the use of unnatural substrates in such reactions.

A triple alkoxo bridged dinuclear cobalt(III) complex mimicking phosphatase and showing ability to degrade organic dye contaminants by photocatalysis

A dinuclear cobalt(III) complex, [(N3)L1CoIIIL2CoIIIL1] (1), has been synthesized and characterized {H2L1 = 2-((1-hydroxybutan-2-ylimino)methyl)-4-bromophenol and HL2 = 2-amino-1-butanol}. Single crystal X-ray diffraction analysis confirmed its structure. Extended supra-molecular architectures were generated in the complex through weak noncovalent interactions. The energetic features of significant supramolecular interactions have been investigated using DFT calculation and further corroborated with NCI plot index computational tool. The complex has been found to mimic the role of phosphatase enzyme efficiently by transforming 4-nitrophenylphosphate to 4-nitrophenolate at room temperature. The reaction follows Michaelis–Menten enzymatic reaction kinetics with turnover numbers of ∼1.4 s−1 in aqueous DMF (98% DMF, v/v) medium. The complex also acts as an efficient photocatalyst for degradation of organic pollutants. Methylene blue (MB), a widely used dye in various industries, was selected as a model pollutant in aqueous media to evaluate the photocatalytic effectiveness of the complex.

Consequences of Heterogeneous Crowding on an Enzymatic Reaction: A Residence Time Monte Carlo Approach.

Translational diffusion of a free substrate in crowded metabolically active spaces such as cell cytoplasm or mitochondrial matrix is punctuated by collisions and nonspecific interactions with soluble/immobile macromolecules/macrostructures in a variety of shapes/sizes. It is not understood how such disruptions alter enzyme reaction kinetics in such spaces. A novel Monte Carlo (MC) technique, “residence time MC”, has been developed to study the kinetics of a simple enzyme–substrate reaction in a crowded milieu using a single immobile enzyme in the midst of diffusing substrates and products. The reaction time lost while the substrate nonspecifically interacts or is transiently trapped with ambient macromolecules is quantified by introducing the residence time “tau”. Tau scales with the size of crowding macromolecules but makes the knowledge of their shape redundant. The residence time thus presents a convenient parameter to realistically mimic the sticky surroundings encountered by a diffusing substrate in heterogeneously crowded physiological spaces. Results reveal that for identical substrate concentration and excluded volume, increase in tau significantly diminished enzymatic product yield and reaction rate, slowed down substrate/product diffusion, and prolonged their relaxation times. A smooth transition from the anomalous subdiffusive motion to normal diffusion at long time limits was observed irrespective of the value of tau. The predictions from the model are shown to be in qualitative agreement with in vitro experimental data revealing the rate of alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate in the midst of 40/500/2000 kDa dextrans. Our findings from the residence time MC model also attempt to rationalize previously unexplained experimental observations in crowded enzyme kinetics literature. Furthermore, major insights to emerge from this study are the reasons why free diffusion of the substrate in crowded physiological spaces is detrimental to enzyme function. It is argued that organized enzyme clusters such as “metabolon” may perhaps exist to regulate the substrate translocation in such sticky physiological spaces to maintain optimal enzyme function. In summary, this work provides key insights explaining why absence of substrate channeling can dramatically slow down enzyme reaction rate in crowded metabolically active spaces.

Combined co-solvent and pressure effect on kinetics of a peptide hydrolysis: an activity-based approach

The application of co-solvents and high pressure has been reported to be an efficient means to tune the kinetics of enzyme-catalyzed reactions. Co-solvents and pressure can lead to increased reaction rates without sacrificing enzyme stability, while temperature and pH operation windows are generally very narrow. Quantitative prediction of co-solvent and pressure effects on enzymatic reactions has not been successfully addressed in the literature. Herein, we are introducing a thermodynamic approach that is based on molecular interactions in the form of activity coefficients of substrate and of enzyme in the multi-component solution. This allowed us to quantitatively predict the combined effect of co-solvent and pressure on the kinetic constants, i.e. the Michaelis constant KM and the catalytic constant kcat, of an α-CT-catalyzed peptide hydrolysis reaction. The reaction was studied in the presence of different types of co-solvents and at pressures up to 2 kbar, and quantitative predictions could be obtained for KM, kcat, and finally even primary Michaelis-Menten plots using activity coefficients provided by the thermodynamic model PC-SAFT.

Kinetic properties of Rhodanese from African locust bean seeds (Parkia biglobosa)

African locust bean (Parkia biglobosa) is a legume which can be processed into condiment. Rhodanese is a sulphurtransferase enzyme. It catalyses the degradation of cyanide to a less toxic metabolite, thiocyanate. Rhodanese purication and biochemical characterization were carried out in this study. The activity of this enzyme from Parkia biglobosa was evaluated and the kinetics of the rhodanese enzyme reaction, including pH and temperature profiles, substrate specificity and effects of metal ions were assessed. Studies on the rhodanese enzyme from other systems have been compared with the results of this study. Rhodanese was isolated from Parkia biglobosa and purified using ammonium sulphate precipitation, CM-Sephadex, and Reactive Blue 2-agarose column chromatography. The purified enzyme has a specific activity of 3.69 RU/mg with a percent yield of 0.20. Km and Vmax values of 7.61 mM and 0.65 RU/ml/min were obtained respectively when KCN was used as a substrate while values of 11.59 mM and 0.57 RU/ml/min was obtained with Na2S2O3. The enzyme showed preference for sodium thiosulphate (Na2S2O3) among different substrates tested namely Mercapto-ethanol (HOCH2CH2SH), Ammonium per sulphate (NH4)2S2O8), Ammonium sulphate (NH4)2SO4), Sodium sulphate (Na2SO4), Sodium metabisulphite (Na2SO5). Optimum temperature and pH of 50?C and 8 were obtained respectively. Chloride salts tested showed little or no inhibitory effect at 1 mM concentration with the exception of HgCl2 and MnCl2. However at 10 mM concentration, the divalent metals; MnCl2 HgCl2, CaCl2, BaCl2 inhibited the enzyme. The present study showed that locust bean seeds possess a cyanide detoxifying enzyme with suitable kinetic properties which renders it safe for consumption.

Mathematical study of a memory induced biochemical system

In this work, to study the effect of memory on a bi-substrate enzyme kinetic reaction, we have introduced an approach to fractionalize the system, considering it as a threecompartmental model. Solutions of the fractionalized system are compared with the corresponding integer-order model. The equilibrium points of the fractionalized system are derived analytically. Their stability properties are discussed from numerical aspect. We determine the changes of the substances due to the changes of &#x201C memory effect &#x201D. The effect is discussed critically from the perspective of product formation. We have also analyzed the memory induced system with a control measure in view of optimizing the product. Our numerical result reveals that the solutions of the fractionalized system, when it is free from memory, are in good agreement with the integer-order system. It is noticed that the effect of memory influences the reaction in the forward direction and assists in yielding the product more quickly. However, an extensive use of memory makes the system slower, but introduction of a control input makes the reaction faster. It is possible to overcome the slowness of the reaction due to the undue effect of memory by appropriate use of a control measure.

Phase-plane geometries in coupled enzyme assays

The determination of a substrate or enzyme activity by coupling one enzymatic reaction with another easily detectable (indicator) reaction is a common practice in the biochemical sciences. Usually, the kinetics of enzyme reactions is simplified with singular perturbation analysis to derive rate or time course expressions valid under the quasi-steady-state and reactant stationary state assumptions. In this paper, the dynamical behavior of coupled enzyme catalyzed reaction mechanisms is studied by analysis of the phase-plane. We analyze two types of time-dependent slow manifolds – Sisyphus and Laelaps manifolds – that occur in the asymptotically autonomous vector fields that arise from enzyme coupled reactions. Projection onto slow manifolds yields various reduced models, and we present a geometric interpretation of the slow/fast dynamics that occur in the phase–planes of these reactions.

Enzymatically Disulfide-Crosslinked Chitosan/Hyaluronic Acid Layer-by-Layer Self-Assembled Microcapsules for Redox-Responsive Controlled Release of Protein.

Disulfide-crosslinked hollow polyelectrolyte microcapsules composed of thiolated chitosan (CS-SH) and hyaluronic acid (HA-SH) were prepared by combining the layer-by-layer (LBL) technique and horseradish peroxidase (HRP)-mediated oxidative cross-linking reaction in mild conditions. FITC-dextran-doped CaCO3 microspheres were used as template core and removed after LBL depositing CS-SH and HA-SH on the surface. The disulfide-crosslinked (CS/HA) microcapsules were readily fabricated by HRP-mediated oxidative coupling of the thiol groups in CS/HA shell layer in the presence of HRP (10 units/mL) and Tyramine hydrochloride (Tyr, 35 mmol/L). The kinetics of enzymatic disulfide-crosslinking reaction was investigated through the real-time monitoring of the consumption of thiol groups by UV absorption spectra. It found that the formation of disulfide linkages by the enzymatic thiol oxidation reaction showed a gradual acceleration. The disulfide-crosslinked CS/HA hydrogel were rapidly formed in gelation time between approximately 17 and 30 min, which were dependent on the concentrations of HRP and Tyr. The disulfide linkages endowed the microcapsule-enhanced physical stability and low permeability under physiological conditions and redox-responsive degradability in reducing environments. The structural stability of disulfide-crosslinked (CS/HA) microcapsules was visualized by confocal laser scanning microscopy in phosphate-buffered saline containing 5.0 mmol/L dithiothreitol (DTT) to evaluate the redox-responsive disassembly process. Redox-responsive controlled release of encapsulated FITC-dextran from the disulfide-crosslinked (CS/HA) microcapsules were obtained. The release profiles of FITC-dextran could be manipulated by controlling the shell thickness and the concentration of DTT. The conformational stability analyses and more than 94% esterase activity of released bovine serum albumin (BSA) from (CS/HA) microcapsules conformed that the structural integrity and bioactivity were well preserved during the encapsulation and release process. The microcapsules exhibited excellent cytocompatibility for HEK 293 cells up to a concentration of 1.0 mg/mL. The microcapsules efficiently delivered loaded FITC-BSA into HeLa cells and released the protein in the reducing cytosol. This study proposed a novel approach for producing disulfide-crosslinked microcarriers for intracellular delivery and redox-responsive controlled release of protein.

Polydopamine functionalized nanoporous graphene foam as nanoreactor for efficient electrode-driven metabolism of steroid hormones

Understanding the enzymatic reaction kinetics of cytochrome P450 (CYP) within confined spaces is essential for the development of efficient CYP bioreactors in vitro. Herein, a facile electrochemical enzymatic nanoreactor (CYP3A4/PNGFs) is constructed by immobilizing CYP3A4 inside polydopamine (PDA) modified nanoporous graphene foams (PNGFs) whose pore diameter is controllable by the template of silica spheres. Taking advantages of the unique 3-D structure of NGFs and the ideal crosslinking agent of PDA, CYP3A4 is easily absorbed into PNGFs with high stability and bioactivity. Meanwhile, the enzymatic activity of CYP3A4 can be effectively regulated by changing the pore diameter of PNGFs. At the pore diameter of about 65 nm, CYP3A4 shows the highest affinity to testosterone with the Michaelis-Menten constant (Kmapp) of 110.70 ± 18 μM and the relatively higher reaction rate with the Imax value of 11.85 ± 1.20 nA, which is due to the maintained native configuration and larger amount of immobilized enzyme. Furthermore, the constructed CYP3A4/PNGF-1 nanoreactor is successfully applied to the metabolism of three steroid hormones (testosterone, estrone and progesterone), and the order of the calculated Kmapp values is consistent with literature. The proposed enzymatic reactor provides a new strategy for the efficient imitation of the natural P450 metabolic pathway at low cost, and has potential applications in fast steroid hormone metabolism assays, drug development and toxicity screening.