Amperometric enzyme electrode for the determination of aspartate aminotransferase and alanine aminotransferase in serum

Glutamate oxidase (E.C. 1.4.3.7) was immobilized at a platinized activated carbon electrode and the enzyme electrodes were used for the amperometric determination of L-glutamate in a stirred aqueous solution by the electrochemical detection of enzymically produced hydrogen peroxide at + 320 mV vs. Ag/AgCl. A linear calibration graph was obtained between 2 μM and 2 mM with a steady-state response time of 1 min. The glutamate oxidase electrode was subsequently applied to the measurement of aspartate aminotransferase (AST) (E.C. 2.6.1.1) and alanine aminotransferase (ALT) (E.C. 2.6.1.2) in serum. The performance of the electrode was compared with that of techniques used in the hospital diagnostic laboratory. The responses of the enzyme electrode to AST and ALT activities were linear over the clinically relevant range (5-500 U l ^?1), and correlated well (r=0.99) with the methods used for routine clinical analysis.     

Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase

The mechanisms of pyridoxal 5'-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine C(α)-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of C(α)-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the C(α)-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C(α)-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a C(α)-H pK(a) that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.     

Separation of aspartate aminotransferase from albumin on substituted agaroses

The affinity of aspartate aminotransferase to its inhibitors coupled to Sepharose 4 B was tested. The affinity was measured as retardation of the enzyme compared to “inert” bovine serum albumin. Carboxylic ligands of citrate and 2-oxoglutarate bound to aminoethyl-Sepharose were the best of those tested in separation of the proteins. Because the ligands were not essentially hydrophobic and because it was shown that ion-exchange is not significant in the elution conditions used, it was suggested that the separation is based on the recognition of substrate or effector by the enzyme.     

Quantitative evaluation of protein recoveries in two-dimensional electrophoresis with immobilized pH gradients

In this study, metabolically radiolabeled Escherichia coli cell extracts were used to systematically evaluate protein recoveries at each step of two-dimensional (2-D) electrophoresis and using different sample application methods. Sample application using sample cups resulted in better protein recovery compared with sample loading by rehydration when the Multiphor system was used. At least 50% or more of an E. coli extract was lost when high protein amounts (500 microg) were loaded by rehydration using this system, which employs separate holders for rehydration and isoelectric focusing (IEF). In contrast, when the IPGphor system was used, rehydration sample loading consistently yielded the highest overall protein recoveries. These improved protein recoveries were due to integration of rehydration and electrophoretic separation in a single unit. Even at high protein loads (500 microg), less than 15-20% of the proteins were lost when proteins were loaded by rehydration using sample buffer containing 2% carrier ampholytes in the ceramic immobilized pH gradient (IPG) strip holders used for both rehydration and IEF. Regardless of the loading conditions used, carrier ampholytes in the sample buffer increased protein recoveries. Use of thiourea did not significantly affect protein recoveries but did improve protein resolution in 2-D gels as expected. In summary, these results show the best protein recoveries are obtained for all protein loads when samples are applied to IPG strips during rehydration using a single device for both rehydration and IEF. In contrast, the poorest recoveries are obtained when rehydration and IEF are performed in separate devices, and losses increase dramatically with increasing protein loads using this approach.     

Capillary electrophoresis with electrochemiluminescence detection for measurement of aspartate aminotransferase and alanine aminotransferase activities in biofluids

A new sensitive assay for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in biofluids was developed, based on the separation and detection of alanine, glutamate, and aspartate using capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The three amino acids were separated in 5 mM phosphate of pH 2.1 as background electrolyte, and detected on a 500 microm platinum disk electrode at 1.2V (versus Ag/AgCl) in the presence of 10 mM tris(2,2'-bipyridyl)ruthenium(II) dissolved in 80 mM phosphate of pH 10.5. A mass detection limit of 37.3 fmol (or 81.5 fmol) for glutamate, corresponding to the product in the enzyme reaction catalyzed by 1.24 x 10(-9)U AST (or 2.72 x 10(-9)U ALT) in a 30 min reaction period, was achieved. This assay was applied to investigate the cytotoxicity effect of ethanol on HepG2 cells and differentiating nonalcoholic steatohepatitis (NASH) from alcoholic liver disease, indicating that the technique is promising for the application in the cell biological and clinical fields.     

Small-angle X-ray scattering studies of the open and closed conformations of aspartate aminotransferase

Summary Aspartate aminotransferase was investigated by X-ray small-angle scattering. A small difference was found between the open (active) and the closed (liganded) conformation of the enzyme. The results were compared with X-ray crystallography data.

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Untersuchungen zur Röntgenkleinwinkelstreuung der offenen und geschlossenen Konformation von Aspartat-Aminotransferase

Zusammenfassung Aspartat-Aminotransferase wurde mittels Röntgenkleinwinkelstreuung untersucht. Ein kleiner Unterschied zwischen der offenen (aktiven) und der geschlossenen (ligandierten) Konformation wurde gefunden. Die Ergebnisse wurden mit Röntgenkristallstrukturdaten verglichen.

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Abbreviations AspAT aspartate aminotransferase     

Unfolding of truncated and wild type aspartate aminotransferase studied by size-exclusion chromatography

The reversible unfolding of globular proteins with increasing concentration of guanidinium chloride (GuCl) can be analysed by size-exclusion chromatography, because the hydrodynamic volume of the proteins increases during unfolding. The dimeric enzyme aspartate aminotransferase (AAT) shows an uncoupled dissociation of the identical subunits followed by the unfolding of the monomers. During the monomer unfolding formation of an intermediate is observed. A monomeric mutant of AAT unfolds with a similar shape of the unfolding transition phase, but is less stable, as shown by a shift of the transition mid-point from 1.7 M GuCl for the wild type to 1.3 M GuCl for the mutant.     

Comparison of recoveries of trichloroethylene from charcoal tubes and thermally-desorbable personal monitors

Solvent-extractable NIOSH charcoal tubes and thermally-desorbable Simtec Adsorba porous polymer monitors for trichloroethylene are compared for recovery efficiencies. The latter monitors are shown to have better recovery efficiencies with less variability than the charcoal monitors. Methods of distribution-free statistical evaluation are outlined for comparing monitors so that reliable conclusions can be drawn.     

Effects of carbodiimides as coupling agents on the stability of immobilized aldolases

Pig muscle aldolase was covalently attached to a polyacrylamide support possessing carboxylic functional groups activated by various water-soluble disubstituted carbodiimides. The stability and the degree of microheterogeneity of the immobilized aldolases were shown to depend on the structure of the carbodiimide used as coupling agent.     

Capillary electrophoresis mass spectrometry coupling with immobilized enzyme electrospray capillaries

Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.     

Comparison of immobilized enzyme reactors for flow-injection systems

Straight, coiled, beaded, and packed-bed reactors containing immobilized glucose oxidase or l-(+)-lactate dehydrogenase were compared in terms of reaction rate, sensitivity, sample throughput, and sample dispersion. The enzymes were covalently attached to the inside walls of 5.0-cm long, 1.12-mm i.d. nylon tubes, and the resulting reactors were tested in a flow-injection system. The beaded enzymatically-active reactors (BEARs) were filled with solid glass beads of 0.5-mm or 1.0-mm diameter. Reactors with the larger beads had 2–4 times the activity, twice the sensitivity, and better throughput than the open reactors; they also minimized the physical and chemical contributions to dispersion. Packed-bed reactors were superior in the lactate determination, but the beaded reactors were better for the determination of glucose. With BEARs containing 1.0-mm beads, glucose was determined in the range 10–800 μM with a conversion efficiency of 0.056 mol of product per mole of substrate; for lactate, the range was 8–64 μM with a conversion efficiency of 0.13 mol mol^?1.     

Characterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS

The preparation and characterization of a miniaturized trypsin reactor using on-line coupling with an ESI-TOF mass spectrometer are described. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was covalently immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith prepared in a 75 microm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multistep reaction. For on-line protein digestion-MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on-line flow through the system with flow rates of 50-300 nL/min. The resulting protein consumption was in the atto- to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 25 degrees C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 37 degrees C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in-solution protocols.     

Comparison of recoveries of inogranic and organic incorporated75Se by four mineralization methods of biological material

The recovery of selenium⁷⁵Se added as selenite to human blood and to mixed food, and the recovery of biologically incorporated⁷⁵Se from different rat tissues were determined by using four mineralization methods. The recoveries of⁷⁵Se after dry ashing /HNO₃, Mg/NO₃/₂/ and after three wet digestion methods — 1. HNO₃, HClO₄ 2. HNO₃, HClO₄, H₂SO₄ 3. HNO₃, HClO₄, MgCl₂ were as follows: 50–106%, 96–99%, 92–99% and 97–100%, resp. Losses of⁷⁵Se in wet digestion /HNO₃, HClO₄/ were observed at the end of the procedure, when an excess of acids was evaporated. The addition of MgCl₂ to the digestion mixture prevented the escape of⁷⁵Se and thus permitted the total evaporation of the digest without any loss of selenium.     

Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports

Enzymes were first immobilized on inorganic supports through silane coupling agents over 25 yr ago. Since that initial report, literally hundreds of laboratories have utilized this methodology for the immobilization of enzymes, antigens, antibodies, receptors, and other high and low mol wt compounds. Today silane coupling is one of the commonly used techniques in the arsenal of the biochemist for the binding of material of all sorts to inorganic surfaces. Inorganic materials come in a variety of shapes, sizes, and characteristics. Today silane coupling is one of the most used coupling methods for the preparation of biosensing devices. Sol-gel entrapped enzymes are also produced by the application of silane technology by the polymerization of the silane to form glass-like materials with entrapped protein. This review will discuss the general preparation and characterization of silane coupled proteins with special emphasis on enzymes and describe in detail the actual methods for the silanization and specific chemical coupling of proteins to the silanized carrier.     

An N⋯H⋯N low-barrier hydrogen bond preorganizes the catalytic site of aspartate aminotransferase to facilitate the second half-reaction

Pyridoxal 5′-phosphate (PLP)-dependent enzymes have been extensively studied for their ability to fine-tune PLP cofactor electronics to promote a wide array of chemistries. Neutron crystallography offers a straightforward approach to studying the electronic states of PLP and the electrostatics of enzyme active sites, responsible for the reaction specificities, by enabling direct visualization of hydrogen atom positions. Here we report a room-temperature joint X-ray/neutron structure of aspartate aminotransferase (AAT) with pyridoxamine 5′-phosphate (PMP), the cofactor product of the first half reaction catalyzed by the enzyme. Between PMP N_SB and catalytic Lys258 Nζ amino groups an equally shared deuterium is observed in an apparent low-barrier hydrogen bond (LBHB). Density functional theory calculations were performed to provide further evidence of this LBHB interaction. The structural arrangement and the juxtaposition of PMP and Lys258, facilitated by the LBHB, suggests active site preorganization for the incoming ketoacid substrate that initiates the second half-reaction.

The neutron structure of pyridoxal 5′-phosphate-dependent enzyme aspartate aminotransferase with pyridoxamine 5′-phosphate (PMP) reveals a low-barrier hydrogen bond between the amino groups of PMP and catalytic Lys258, preorganizing the active site for catalysis     

A QM/MM simulation study of transamination reaction at the active site of aspartate aminotransferase: Free energy landscape and proton transfer pathways

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5^' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α -carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.     

Comparison of catalytic properties of free and immobilized cellobiase Novozym 188

The enzyme cellobiase from Novo was immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde method with protein and activity yields of 67 and 13.7%, respectively. The activity of the free enzyme (FE) and immobilized enzyme (IE) was determined with 2 g/L of cellobiose, from 40 to 75°C at pH 3.0–7.0 for FE and from 40 to 70°C at pH 2.2–7.0 for IE. At pH 4.8 the maximum specific activity for the FE and IE occurred at 65°C: 17.8 and 2.2 micromol of glucose/(min·mg of protein), respectively. For all temperatures the optimum pH observed for FE was 4.5 whereas for IE it was shifted to 3.5. The energy of activation was 11 kcal/mol for FE and 5 kcal/mol for IE at pH 4.5–5, showing apparent diffusional limitation for the latter. Thermal stability of the FE and IE was determined with 2 g/L of cellobiose (pH 4.8) at temperatures from 40 to 70°C for FE and 40 to 75°C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures up to 55°C. The IE has shown higher stability, retaining its activity in the sametest up to 60°C. Half-life experimental results for FE were 14.1, 2.1, and 0.17 h at 60, 65, and 70°C, respectively, whereas IE at the same temperatures had half-lives of 245, 21.3, and 2.9 h. The energy of thermal deactivation was 80.6 k cal/mol for the free enzyme and 85.2 k cal/mol for the IE, suggesting stabilization by immobilization.     

Comparison of various coupling reagents in solid-phase aza-peptide synthesis

Aza-peptides are promising drug leads, however extensive study of their properties is hampered by low yielding aza-peptide bond formation during conventional Fmoc SPPS. The kinetics of aza-peptide bond formation in the model peptide H-Ala-AzAla-Phe-NH₂ was compared with various conventional amino acid activators. The reaction rates and yields were dependent on the activator structure. The reaction time of aza-peptide formation using oxyma-based agents was approximately 30 times longer than in typical peptide synthesis. Therefore, new activators are required to increase the reactivity of the activated amino acid to achieve effective acylation of the semicarbazide moiety during aza-peptide bond formation.     

Improving the recoveries of unstable N-chloramines determined by liquid chromatography-postcolumn electrochemical detection

Liquid chromatographic (LC) measurement of individual N-chloramines, which are key byproducts of wastewater and drinking water chlorination, could lead to more effective control of water disinfection. Such measurements are challenging because of analyte instability. A detector selective for N-chloramines is constructed based on postcolumn derivatization with iodide followed by reductive detection of the iodine product at a glassy carbon electrode. In flow injection (FIA) mode, the detector gives identical responses for a test set of four chemically diverse N-chloramines. In the LC mode, losses of the test compounds are observed when LC and FIA responses are compared and quantitated by introducing a relative response factor (RRF). Using the RRF, N-chloramine recoveries are evaluated as a function of multiple LC separation parameters. The highest recoveries are obtained using a reversed-phase (C18) column with an acetonitrile mobile phase and a pH 7.02 aqueous phosphate buffer. With these conditions, linear calibration curves are obtained for all test N-chloramines. The detection limits obtained are in the low 10(-7)-mol/L range, which is nearly tenfold better than previously reported and 10-1000-fold lower than total residual chlorine concentrations typically found in disinfected water and wastewater.