Inclusion of Polyphenol Oxidase Substrates in β-Cyclodextrin: A 1 H-NMR Study

A ¹H-NMR study of the interactionsbetween -cyclodextrin (-CD) and included phenolic molecules (chloragenic acid and caffeic acid ) in aqueous medium is reported. The results confirm that inclusion occurs. Data analysis by the continuous variation method shows that all the complexes have 1 : 1 stoichiometries. Values for the apparent association constants of the inclusion compounds are estimated and compared with previously reported values.     

Probing the Conformational Stability of Two Different Copper Proteins: A Dynamic Fluorescence Study on Azurin and Ascorbate Oxidase

Tryptophan fluorescence is extremely useful to monitor structural conformational transitions in proteins. Denaturant-induced unfolding of azurin and ascorbate oxidase has been studied by dynamic fluorescence measurements in the frequency domain and the results have been interpreted in terms of continuous distribution of lifetimes. The data add new information on the unfolding mechanism that was previously analyzed by steady-state emission spectroscopy. In particular, the existence of multiple, parallel unfolding pathways may be envisaged and correlated, in both cases, to the two protein structures. The effect of metal depletion has been also characterized by fluorescence lifetime measurements. In the case of azurin, a monomeric protein, the data demonstrate that copper removal yields a totally different unfolding pathways with respect to the holo protein, indicating that metal ion plays a fundamental structural role in the wild type, native protein. In the case of ascorbate oxidase a dimer of 140 kDa, only minor effects have been detected by copper removal. However, the analysis of the fluorescence decay in presence of different amounts of guanidinium hydrochloride gives new important insights on the unfolding intermediates. In particular the data support the hypothesis of a partial exposure of an outer layer of dimer at intermediate denaturant concentration. This ability of dynamic fluorescence to pinpoint the presence of structural micro-heterogeneity in the unfolding pathways of proteins demonstrates the greater power of this technique compared to the most commonly used steady-state measurements.     

Models for the active site in galactose oxidase: Structure, spectra and redox of copper(II) complexes of certain phenolate ligands

Galactose oxidase (GOase) is a fungal enzyme which is unusual among metalloenzymes in appearing to catalyse the two electron oxidation of primary alcohols to aldehydes and H₂O₂. The crystal structure of the enzyme reveals that the coordination geometry of mononuclear copper(II) ion is square pyramidal, with two histidine imidazoles, a tyrosinate, and either H₂O (pH 7.0) or acetate (from buffer,pH 4-5) in the equatorial sites and a tyrosinate ligand weakly bound in the axial position. This paper summarizes the results of our studies on the structure, spectral and redox properties of certain novel models for the active site of the inactive form of GOase. The monophenolato Cu(II) complexes of the type [Cu(L1)X][H(L1) = 2-(bis(pyrid-2-ylmethyl)aminomethyl)-4-nitrophenol and X⁻ = Cl⁻ 1, NCS⁻ 2, CH₃COO⁻ 3, ClO₄ ⁻ 4] reveal a distorted square pyramidal geometry around Cu(II) with an unusual axial coordination of phenolate moiety. The coordination geometry of 3 is reminiscent of the active site of GOase with an axial phenolate and equatorial CH₃COO⁻ ligands. All the present complexes exhibit several electronic and EPR spectral features which are also similar to the enzyme. Further, to establish the structural and spectroscopic consequences of the coordination of two tyrosinates in GOase enzyme, we studied the monomeric copper(II) complexes containing two phenolates and imidazole/pyridine donors as closer structural models for GOase. N,N-dimethylethylenediamine and N,N’-dimethylethylenediamine have been used as starting materials to obtain a variety of 2,4-disubstituted phenolate ligands. The X-ray crystal structures of the complexes [Cu(L5)(py)], (8) [H₂(L5) = N,N-dimethyl-N’,N’-bis(2-hydroxy-4-nitrobenzyl) ethylenediamine, py = pyridine] and [Cu(L8)(H₂O)] (11), [H₂(L8) = N,N’-dimethyl-N,N’-bis(2-hydroxy-4-nitrobenzyl)ethylenediamine] reveal distorted square pyramidal geometries around Cu(II) with the axial tertiary amine nitrogen and water coordination respectively. Interestingly, for the latter complex there are two different molecules present in the same unit cell containing the methyl groups of the ethylenediamine fragmentcis to each other in one molecule andtrans to each other in the other. The ligand field and EPR spectra of the model complexes reveal square-based geometries even in solution. The electrochemical and chemical means of generating novel radical species of the model complexes, analogous to the active form of the enzyme is presently under investigation.     

Microbiosensor based on glucose oxidase and hexokinase co-immobilised on platinum microelectrode for selective ATP detection

ATP determination is of great importance since this compound is involved in a number of vital biological processes. To monitor ATP concentration levels, we have developed a microbiosensor based on cylindrical platinum microelectrode, covered with a layer of poly-m-phenylendiamine (PPD), and layer of co-immobilised glucose oxidase and hexokinase. Conditions for biosensor measurement of ATP (pH, Mg²⁺ and substrates concentration) in vitro and microbiosensor characteristics such as sensitivity, selectivity, reproducibility, storage stability were studied and optimized. Under optimal conditions the microbiosensor can measure ATP concentrations down to a 2.5 μM detection limit with response time about 15 s. Interferences by electroactive compounds like biogenic amines and their metabolites, ascorbic acid, uric acid and l-cystein are rejected in general by the PPD layer. The microbiosensor developed is insensitive to ATP analogues (or substances with similar structure), such as ADP, AMP, GTP and UTP, too. It can be used for ATP analysis in vitro in the reactions consuming or producing macroergic triphosphate molecules to study kinetics of the process and in drug design concerning development of inhibitors specific to target kinases and others target enzymes.     

Electrochemical Kinetic Characterization of Redox Mediated Glucose Oxidase Reactions: A Simplified Approach

One of the main drawbacks affecting first‐generation electrochemical biosensors in the analysis of real matrices is the interference of electroactive species present in the sample under investigation. Several approaches have been attempted to overcome this problem in the past ten years but the best results were achieved by using mediated based electrochemical biosensors. Despite this, the kinetic of the redox mediators‐enzymatic proteins interaction has not been studied deeply enough. In this work we have developed a theoretical‐methodological approach for the characterization of the kinetic of interaction between redox enzymes and substrates and/or redox mediators. Particularly, the interaction of glucose oxidase (GOx) with several commercially available redox mediators has been studied by means of amperometry and cyclic voltammetry. The main kinetic parameters for different mediators were exploited and discussed with the aim of finding the best mediator for a glucose biosensor to be used on real samples.     

Development of a bienzyme reactor sensor system for the determination of ornithine

A bienzyme reactor sensor system with amperometric detection was developed for the determination of ornithine. The system based on the immobilized enzymes (ornithine carbamyl transferase and pyruvate oxidase) consisted of a buffer tank, a peristaltic pump, an enzyme reactor, an oxygen electrode and a recorder. Then, 0.1 M MOPS buffer, containing pyruvic acid (0.5 mM) and carbamyl phosphate (0.5 mM), was continuously transferred into the system at 35 °C. Phosphate ion was formed enzymatically by transformation of ornithine in the presence of carbamyl phosphate. Pyruvate oxidase is activated by the presence of phosphate. Therefore, ornithine was determined from the oxygen consumed upon oxidation of pyruvic acid catalyzed by pyruvate oxidase in the presence of phosphate ion. The limit of detection was 0.05 mM and the response was linear to 3 mM (R²=0.9905). The variation coefficients were 4.9 (n=15) and 3.9% (n=15) for 1.1 and 3.0 mM standard ornithine, respectively. Good comparative results (R²=0.9238) were observed between ornithine contents in prawn muscle determined by the proposed system and by the HPLC. One assay was completed within 4 min. The immobilized enzymes were stable for 2 months at 4 °C and more than 150 samples could be continually determined using this enzyme reactor.     

Synthesis of Biologically Active[4-(4-Bromophenyl)-2-thiazolyl]hydrazones and their D-Galactose Derivatives

Benzaldehyde [4‐(4‐bromophenyl)thiazol‐2‐yl]hydrazones 5a – 5d were prepared by reacting the thiosemicarbazones 2a – 2d with 2,4′‐dibromoacetophenone ( 1 ) in absolute ethanol. Acetylation of 5a and 5b with Ac₂O/Py at room temperature gave the N‐acetyl derivatives 6a and 6b . 4‐Methyl‐2‐pentanone/cyclopentanone [4‐(4‐bromo‐phenyl)thiazol‐2‐yl]hydrazones ( 8a ) and ( 8b ) were similarly obtained from the reaction of 1 with the thiosemicarbazones 7a and 7b , respectively. Cyclization of D‐galactose thiosemicarbazone ( 9 ) and its tautomers 10 and 11 with 1 afforded an equilibrium mixture of the acyclic 2‐thiazolylhydrazone 12 , together with its respective cyclic galactosyl derivatives 13 and 14 , whose structures were studied by using ¹H and ¹³C NMR spectra. The antimicrobial activity of the synthesized thiazole derivatives was evaluated in vitro by using an agar diffusion technique, and some of these compounds showed potential activity against Candida albicans.     

Real‐Time Monitoring of Mass‐Transport‐Related Enzymatic Reaction Kinetics in a Nanochannel‐Array Reactor

To understand the fundamentals of enzymatic reactions confined in micro‐/nanosystems, the construction of a small enzyme reactor coupled with an integrated real‐time detection system for monitoring the kinetic information is a significant challenge. Nano‐enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real‐time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass‐transport‐related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano‐enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50 μL min^?1), the enzymatic reaction kinetics became the rate‐determining step. This change resulted in the decrease in the conversion efficiency of the nano‐enzyme reactor and the apparent Michaelis–Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis.     

Amine-terminated organosilica nanosphere functionalized prussian blue for the electrochemical detection of glucose

A new glucose biosensor had been developed by immobilizing positively charged gold nanoparticles (PGNs) on organosilica nanosphere functionalized prussian blue (OSiFPB)-modified gold electrode. The OSiFPB compound could not only effectively prevent the leakage of the PB mediator during measurements, but also easily form stable film on the electrode surface with efficient redox-activity and excellent conductivity. Furthermore, with the negatively charged surface of OSiFPB, this film could be used as an interface to adsorb the PGNs, which provided a congenial microenvironment for adsorbing biomolecules and decreased the electron-transfer impedance. So, with glucose oxidase as a model biomolecular, the proposed sensor showed rapid and highly sensitive amperometric response to glucose and this immobilization approach effectively improved the stability of the electron-transfer mediator. This work would be promising for construction of biosensor and bioelectronic devices.     

Glucose concentration determination based on silica sol-gel encapsulated glucose oxidase optical biosensor arrays

Optical biosensor arrays for rapidly determining the glucose concentrations in a large number of beverage and blood samples were developed by immobilizing glucose oxidase (GOD) on oxygen sensor layer. Glucose oxidase was first encapsulated in silica based gels through sol-gel approach and then immobilized on 96-well microarrays integrated with oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)₃Cl₂). The oxidation reaction of glucose by glucose oxidase could be monitored through fluorescence intensity enhancement due to the oxygen consumption in the reaction. The luminescence changing rate evaluated by the dynamic transient method (DTM) was correlated with the glucose concentration with the wide linear range from 0.1 to 5.0 mM (Y = 13.28X − 0.128, R = 0.9968) and low detection limit (0.06 mM). The effects of pH and coexisting ions were systemically studied. The results showed that the optical biosensor arrays worked under a wide range of pH value, and normal interfering species such as Na⁺, K⁺, Cl⁻, PO₄³⁻, and ascorbic acid did not cause apparent interference on the measurement. The activity of glucose oxidase was mostly retained even after 2-month storage, indicating their long-term stability.