Scanning electrochemical microscopy of enzymes immobilized on structured glass-gold substrates

Scanning electrochemical microscopy (SECM) was used to characterize enzyme-modified glass-gold specimens. The exposed gold surface was functionalized with an aminothiol and reacted with carbodiimide-activated glucose oxidase. The specimen surface was examined with SECM, using a 25 μm platinum electrode. Images were acquired showing the topography, electric conductivity, and enzymatic activity of the composite surface. It was found that the hydroxy-groups of the glass surface are as likely to bind to the activated enzyme as the amino-groups on the gold surface.     

Stopped-flow kinetic determination of glucose and lactate with immobilized enzymes

Kinetic procedures are described for the measurement of lactate and glucose with enzymes immobilized in the observation cellof a stopped-flow spectrophotometer. Kinetic data are obtained by two-point or multipoint methods with data acquisition over any time range desired. Glucose was quantified in a fixed-time mode with a linear range of 0–10 mM. Lactate was quantified in the range 0–50 μM by obtaining the slope of the absorbance/time profile between 10 and 20 s after each reaction was initiated. Results for these two substrates in control sera are presented.     

Affinity of trypsin for amidine derivatives immobilized on dextran-coated silica supports.

The pancreas contains two very analogous enzymes: trypsin and chymotrypsin. These two enzymes are very similar in their physicochemical characteristics and are therefore quite difficult to separate by classical purification procedures. They constitute a good model for affinity chromatography. It was previously demonstrated that amidine derivatives are able to interact strongly and specifically with these serine proteases and are often used as ligand in affinity chromatography. To understand the trypsin interaction mechanism, we synthesized different amidines and immobilised them with or without spacer arm on silica beads previously coated by dextran substituted with a calculated amount of positively charged diethylaminoethyl functions, in order to minimize the non-specific interactions of silanol groups of the silica material. First the affinity constant and the adsorption capacity of these supports for trypsin were determined in batch procedures, then they were used in affinity chromatography. The effects of ionic strength, pH and competitive inhibitors on proteins desorption were also studied. Last, to demonstrate the importance of passivation, the chromatographic performances of dextran-coated silica phases and a commercial support grafted with the same amidine were compared.     

Electrochemistry of immobilized redox enzymes: kinetic characteristics of NADH oxidation catalysis at diaphorase monolayers affinity immobilized on electrodes

In the class of NADH:acceptor oxidoreductases, the diaphorase from Bacillus stearothermophilusis a particularly promising enzyme for sensing NADH, and indirectly a great number of analytes, when coupled with a NAD-dependent dehydrogenase as well as for the design of mono- and multienzyme affinity sensors. The design and rational optimization of such systems require devising immobilization procedures that prevent dramatic losses of the enzymatic activity and a full kinetic characterization of the immobilized enzyme system. Two immobilization procedures are described, which involve recognition of the biotinylated diaphorase by a monolayer of neutravidin adsorbed on the electrode surface either directly or through the intermediacy of a monolayer of biotinylated rabbit immunoglobulin. Thorough kinetic characterization of the two systems is derived from cyclic voltammetric responses. A precise estimate of the enzyme coverages is obtained after comparing the enzyme kinetics of the immobilized and the homogeneous system.     

Further characterization and kinetic parameter determination of a milk-clotting protease fromMucor bacilliformis

Further characterization of an aspartyl protease fromMucor bacilliformis with milk-clotting activity was performed. An extinction coefficient, ε_{278 cm} = 1.61 mL/mg/cm, a molecular mass of 35,400 Da and a pI of 5.2 were determined. Proteolytic activity and kinetic parameters were evaluated by using the hexapeptide Leu-Ser-pNO₂-Phe-Nle-Ala-Leu-OMe as the substrate. The effect of pH and temperature on peptide cleavage, as well as protease heat stability, was determined. Such properties, taken as a whole, indicate that theM. bacilliformis protease can be considered a potential substitute for bovine chymosin in cheese manufacture.     

Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken.

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23,500, 25,700 and 25,000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and k(cat)/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.     

Microscale characterization of the structure-activity relationship of a heparin-binding glycopeptide using affinity capillary electrophoresis and immobilized enzymes.

A heparin-binding glycopeptide (T3) from human serum amyloid P component was characterized by taking advantage of two important features of capillary electrophoresis: the low sample consumption and the possibility of doing on-line binding studies. Incubations with neuraminidase and proteolytic enzymes were carried out with enzymes immobilized on paramagnetic microbeads. Affinity capillary electrophoresis subsequently was used to characterize T3 and its fragments with respect to heparin binding. We find that an intact glycan moiety makes the C-terminal part of T3 relatively resistant to chymotryptic clevage. This protection is lost upon desialylation. Also, the C-terminus of T3 is involved in heparin binding while the N-terminal part of the molecule has no appreciable binding activity. The micromethods presented here make it feasible to perform structure-function studies even on the small amounts of analytes that are typically available when working with glycopeptides from natural sources.     

Automatic flow methodology for kinetic and inhibition studies of reactions with poorly water-soluble substrates in ionic liquid systems

In the present work an automatic generic tool, based on sequential injection analysis (SIA) for kinetic and inhibition studies of reactions with poorly water-soluble compounds in ionic liquid (IL)-containing systems, is described.The oxidation of the poorly water-soluble phenolic compound, caffeic acid, catalyzed by the mushroom tyrosinase, in different 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF₄])/buffer mixtures as reaction media, was investigated. This determination was based on measuring depletion rate of the substrate caffeic acid at its maximum wavelength (λ_max 311 nm).The influence of several parameters such as substrate and enzyme concentration, temperature, pH, delay times and measurement periods on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected.The obtained results showed that tyrosinase was active in oxidising caffeic acid in this water-miscible IL and the presence of an impaired tyrosinase activity with increase in [bmim][BF₄] concentration as an increase in the apparent Michaelis–Menten constant () was observed while the maximum reaction rate () remained fairly constant. The results were compared to those obtained when the assay was performed in water/methanol mixtures under the same conditions to substantiate [bmim][BF₄] as an alternative to conventional organic solvents.Additionally, it was shown that tyrosinase is effectively inhibited by the substrate analogues tested (trans-cinnamic acid and 3,4-dihydroxybenzoic acid) in the IL-containing aqueous system used.     

Selective removal of enzymes from substrate and products. An alternative to immobilization for enzymes acting on macromolecular or solid substrates

A new approach for the control and interruption of enzymatic reactions via selective enzyme immobilization has been developed. The technique was exemplified by the use of three model enzymes with the corresponding macromolecular substrates: α-amylase/starch, trypsin/ insoluble collagen, and alkaline phosphatase/plasmid DNA. Prior to incubation with its substrate, each enzyme was provided withde novo thiol-groups by a two-step reaction involvingN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and DTT. The chemical modification was achieved such that at least 80% of the native enzyme activity was preserved in all cases. In order to interrupt rapidly the reactions in which the enzymes were used, the modified enzyme was immobilized by reaction via its thiol groups on a thiolsulfinate-agarose derivative. The gel-bound enzyme could then be easily removed from unreacted substrate and product by filtration or centrifugation. Comparative studies showed that the immobilized enzymes had much lower activities in the reactions studied than the corresponding soluble ones. The potential for enzyme reuse was also demonstrated with the a-amylase derivatives, which were quantitatively released and eluted in fully active form from the agarose. We have shown that it is possible to achieve practically complete enzyme immobilization in short times and thus to control the progress of the reactions. Because of its simplicity and high efficiency, this approach may represent an interesting alternative for biotechnological processes involving macromolecular or solid substrates.     

Some kinetic parameters and inactivation of catalase immobilized on modified polyvinylalcohol

Polyvinylalcohol crosslinked with terephthaldicarboxaldehyde and was modified with 2-amino-4,6-dichloro-s-triazine. Optimum conditions for immobilization of catalase on modified and gelatine-coated modified polyvinylalcohol were investigated. Activity variations with respect to pH, temperature, stability behavior, andk _m(appl) values were investigated for the native and immobilized catalases. Rate constants for H₂O₂ decomposition and for inactivation of immobilized catalase were determined using a discontinuous batch-type reactor. The influence of H₂O₂ concentration on the catalase inactivation was investigated.     

Flow-injection analysis systems with immobilized enzymes. Improvement of applicability by integration of coupled reactions, separation steps and background correction

The specificity of enzymes is often not sufficient to simultaneously determine two parent substrates in a given matrix. Approaches to enhance the selectivity by applying the principle of an array arrangement to flow-injection analysis (FIA) systems based on several immobilized isoenzymes are successful. Immobilized enzymes and detectors in FIA systems often suffer from interferences from impurities of the matrix. Examples are given which prove that this problem can be overcome by an integrated preseparation of the analyte (pervaporation, electrodialysis) or by correction of the matrix signal based on background subtraction using computerized data accumulation and processing.     

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.     

Green synthesis of Au nanoparticles immobilized on halloysite nanotubes for surface-enhanced Raman scattering substrates

A facile and green route was introduced to synthesize Au nanoparticles immobilized on halloysite nanotubes (AuNPs/HNTs) used for surface-enhanced Raman scattering substrates. The naturally occurring HNTs were firstly functionalized with a large amount of -NH(2) groups by N-(β-aminoethyl)-γ-aminopropyl trimethoxysilane (AEAPTES), which possesses one lone electron pair and will "anchor" Au ions to form a chelate complex. Then, with the addition of tea polyphenols (TP), the Au ions were reduced on the surface of the previously formed Au-NH(2) chelate complex to form AuNPs. Transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) observations indicate that a large amount of AuNPs were synthesized on HNTs. The AuNPs are irregularly spherical and densely dispersed on HNTs and the diameter of the nanoparticles varies from 20 to 40 nm. The interactions between AuNPs and -NH(2) groups were verified by X-ray photoelectron spectroscopy (XPS) and the results showed that the functional groups can "anchor" AuNPs through the chelating effect. The as-prepared AuNPs/HNTs nanomaterials with several nanometers gaps among nanoparticles were used as a unique surface-enhanced Raman scattering substrate, which possessed strong and distinctive Raman signals for R6G, indicating the remarkable enhancement effect of the AuNPs/HNTs.     

Quantitative analysis and characterization of DNA immobilized on gold

We describe the complementary use of X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy to quantitatively characterize the immobilization of thiolated (dT)(25) single-stranded DNA (ssDNA) on gold. When electron attenuation effects are accurately accounted for in the XPS analysis, the relative coverage values obtained by the two methods are in excellent agreement, and the absolute coverage can be calculated on the basis of the XPS data. The evolution of chemically specific spectral signatures during immobilization indicates that at lower coverages much of the DNA lies flat on the surface, with a substantial fraction of the thymine bases chemisorbed. At higher immobilization densities, the (dT)(25) film consists of randomly coiled ssDNA molecules each anchored via the thiol group and at possibly one or two other bases. We use two examples to demonstrate how the quantitative analysis can be applied to practical problems: the effects of different buffer salts on the immobilization efficiency; the immobilization kinetics. Buffers with divalent salts dramatically increase the efficiency of immobilization and result in very high surface densities (>5 x 10(13)/ cm(2)), densities that may only be possible if the divalent counterions induce strong attractive intermolecular interactions. In contrast with previous reports of alkanethiol adsorption kinetics on gold, ssDNA immobilization in 1 M phosphate buffer does not occur with Langmuir kinetics, a result attributable to rearrangement within the film that follows the initial adsorption.     

Mechanism of serine protease action. Ionization behavior of tetrahedral adduct between α-lytic protease and tripeptide aldehyde studied by carbon-13 magnetic resonance

Magnetic resonance techniques have been used to study the ionization behavior of the catalytic triad of the serine protease, α-lytic protease, in the tetrahedral, hemiacetal complex it forms with the aldehyde inhibitor, N-ac-L -ala-L -pro-L -alaninal. Chemical shift, coupling constant and relaxation measurements of a carbon-13 nucleus specifically incorporated in C-2 of the imidazole ring of the single histidine residue of the protein show that, above pH 7, the imidazole ring of the catalytic triad in the enzyme + aldehyde complex is neutral. We suggest, further, that a neutral carboxylic acid group for Asp 102 and an oxyanion for the hemiacetal are most likely to describe the state of ionization of the other groups above pH 7. Around pH 6·25, both the oxyanion and the histidine become protonated in a co-operative process which forces the histidine away from its rigidly localized position as a member of the catalytic triad into a solution-like environment.