Monitoring carbohydrate enzymatic reactions by quantitative in vitro microdialysis

On-line in vitro microdialysis (MD) sampling followed by HPLC separation and UV absorbance detection (HPLC-UV) was used to monitor carbohydrate enzyme systems. Fundamental parameters (i.e., K(m) and V(max)) of hydrolysis reactions of 4-nitrophenyl-beta-D-glucopyranoside, 4-nitrophenyl-beta-d-galactopyranoside, and 4-nitrophenyl-beta-D-xylopyranoside were determined for a model enzyme, almond beta-glucosidase. Accurate quantitation was achieved via internal standard methodology and compared to spectrophotometric data and literature K(m) values, which were found to be 2.6+/-0.5 mM (MD), 2.7+/-0.4 mM (spec), and 2.5 mM (lit), for the substrate 4-nitrophenyl-beta-d-glucopyranoside. A previously unpublished K(m) value for the substrate salicin was also determined by this method. An application is shown for monitoring the glycoside salicin and its hydrolysis product saligenin in a commercially available willow bark product that is used for making tea. This versatile method has far-reaching applications to monitoring a variety of carbohydrates in enzymatic processes without complex sample preparation procedures and without volume loss.     

Impedance analysis of an electrode-separated piezoelectric sensor as a surface monitoring technique for surfactant adsorption on quartz surface

This paper describes the measurement of the kinetics of adsorption of sodium dodecyl sulfate (SDS), an anionic surfactant, onto a quartz surface with a pre-adsorbed layer of Ca2+ as an ion bridge, using an electrode-separated piezoelectric sensor (ESPS). An impedance analysis method was employed to characterize the responses of the ESPS. The impedance and frequency parameters of the ESPS were examined as functions of the conductivity, permittivity, viscosity and density of the liquid. The adsorption process of SDS onto the quartz surface resulted in an increase in both the mass and energy dissipation of the oscillating quartz crystal. The adsorption densities could be estimated by the ESPS method after taking into consideration the effects of surface viscosity and roughness. The adsorption and desorption rate constants of SDS onto the quartz surface were calculated as ka = (88.1 +/- 0.26) mol(-1) L s(-1) and kd = (4.92 +/- 0.53) x 10(-3) s(-1), respectively, based on the Langmuir model. ESPS was shown to be a powerful means of examining anionic surfactant adsorption to the solid/liquid interface.     

Study of the immobilization of alcohol dehydrogenase on Au-colloid modified gold electrode by piezoelectric quartz crystal sensor,cyclic voltammetry,and electrochemical impedance techniques

The immobilization of alcohol dehydrogenase (ADH) on Au-colloid modified gold electrodes has been investigated. Colloidal Au was first self-assembled onto gold electrodes through the thiol groups of an 1,6-hexanedithiol monolayer. Piezoelectric quartz crystal sensor, cyclic voltammetry, and electrochemical impedance techniques were used to investigate the immobilization of ADH on Au colloids. The cyclic voltammogram tends to be more irreversible with increased ADH concentration. In the impedance spectroscopic study, an obvious difference of the electron transfer resistance between the Au-colloid modified electrode and the bare gold electrode was observed. Using the piezoelectric quartz crystal sensor, the Michaelis constant, K(m), and the maximum initial rate, V(max), of the immobilized ADH were estimated as 6.03 x 10(-4) M and 0.63 Hzs (-1), respectively. The binding constant of ADH with nicotinamide adenine dinucleotide (NAD) was also determined as 1.87 x 10(4) M(-1). Experimental results showed that colloidal Au can be used as a biocompatible matrix for enzyme immobilization.     

New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance

The hemoglobin (Hb) released from erythrocytes is a primary nutritive component for many blood-feeding parasites. The aspartic protease cathepsin D is a hemoglobinase that is involved in the Hb degradation process and is considered an interesting target for chemotherapy intervention. However, traditional enzymatic assays for studying Hb degradation utilize spectrophotometric techniques, which do not allow real-time monitoring and can present serious interference problems. Herein, we describe a biosensor using simple approach for the real-time monitoring of Hb hydrolysis as well as an efficient screening method for natural products as enzymatic inhibitors using a quartz crystal microbalance (QCM) technique. Hemoglobin was anchored on the quartz crystal surface using mixed self-assembled monolayers. The addition of the enzyme caused a mass change (frequency shift) due to Hb hydrolysis, which was monitored in real time. From the frequency change patterns of the Hb-functionalized QCM, we evaluated the enzymatic reaction by determining the kinetic parameters of product formation (k_cat). The QCM enzymatic assay using immobilized human Hb was shown to be an excellent approach for screening possible inhibitors in complex mixtures, opening up a new avenue for the discovery of novel inhibitors.     

Kinetics of iterative carbohydrate transfer to polysaccharide catalyzed by chondroitin polymerase on a highly sensitive flow-type 27 MHz quartz-crystal microbalance

Using a highly sensitive flow-type 27?MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97?μM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4) M(-1) s(-1) for GlcA transfer and 3.4×10(4) M(-1) s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.     

Enzymatic kinetics of cellulose hydrolysis: a QCM-D study

The interactions between films of cellulose and cellulase enzymes were monitored using a quartz crystal microbalance (QCM). Real-time measurements of the coupled contributions of enzyme binding and hydrolytic reactions were fitted to a kinetic model that described closely significant cellulase activities. The proposed model combines simple Boltzmann sigmoidal and 1 - exp expressions. The obtained kinetics parameters were proven to be useful to discriminate the effects of incubation variables and also to perform enzyme screening. Furthermore, it is proposed that the energy dissipation of a film subject to enzymatic hydrolysis brings to light its structural changes. Overall, it is demonstrated that the variations registered in QCM frequency and dissipation of the film are indicative of mass and morphological transformations due to enzyme activities; these include binding phenomena, progressive degradation of the cellulose film, existence of residual, recalcitrant cellulose fragments, and the occurrence of other less apparent changes throughout the course of incubation.     

The properties of covalently immobilized trypsin on soap-free P(MMA-EA-AA) latex particles

The covalent immobilization of trypsin onto poly[(methyl methacrylate)-co-(ethyl acrylate)-co-(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55 degrees C and 8.5, both of which were higher than that of the free form. It was found that K(m) (Michaelis constant) was 45.7 mg . ml(-1) and V(max) (maximal reaction rate) was 793.0 microg . min(-1) for immobilized trypsin, compared to a K(m) of 30.0 mg . ml(-1) and a V(max) of 5 467.5 microg . min(-1) for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times.     

Monitoring of mutagenic process with piezoelectric quartz crystal impedance analysis

A novel method for monitoring of mutagenic process of dimethyl sulfate to Salmonella typhimurium strain (TA100) was proposed by using piezoelectric quartz crystal impedance (PQCI) analysis technique. The time courses of responses piezoelectric impedance parameters for a quartz crystal in a culture system were simultaneously obtained and discussed. It was found that the motional resistance variation (DeltaR(m)) increases and frequency shift (Deltaf) of PQC sensor decreases correspondingly during the mutagenic process of the bacteria. These parameters could reflect the variations of viscosity and density of culture system. By fitting DeltaR(m) versus time curves toward Gompertz bacterial growth model, we obtained and discussed the bacterial growth parameters for both normal growth and mutagenic process. The experiments showed that the proposed method could provide real time and multidimensional impedance information to the monitoring of mutagenic process.     

Unexpected stability of aryl beta-N-acetylneuraminides in neutral solution: biological implications for sialyl transfer reactions

A reagent panel comprised of seven aryl beta-D-N-acetylneuraminides was synthesized and then used to probe the mechanisms of nonenzymatic hydrolysis. These reactions proceeded via four independent pathways: (1) acid-catalyzed hydrolysis of the neutral molecule; (2) acid-catalyzed hydrolysis of the anionic form, or its kinetic equivalent spontaneous hydrolysis of the neutral form; (3) spontaneous hydrolysis of the anionic form; and (4) a base-promoted pathway. The pH-independent spontaneous hydrolysis of 4-nitrophenyl alpha-D-N-acetylneuraminide (5) occurs at a rate that is over 100 times faster than that of the corresponding reaction of 4-nitrophenyl beta-D-N-acetylneuraminide (4a). Spontaneous hydrolyses of four aryl beta-D-N-acetylneuraminides displayed a beta(lg) value of -1.24 +/- 0.16 (pH = 8.1, T = 100 degrees C), and at a pH value of 1.0 (50 degrees C), all seven panel members gave a beta(lg) value of 0.14 +/- 0.08. The aqueous ethanolyses of 4a and 5 gave similar products and displayed sensitivity parameters (m) in a standard Winstein-Grunwald analysis of -0.04 +/- 0.01 and +0.23 +/- 0.02, respectively. These results, plus the activation parameters calculated for the spontaneous hydrolyses of the anionic forms of 5 (DeltaH() = 116 +/- 2 kJ mol(-1) and DeltaS = 27 +/- 4 J mol(-1) K(-1)) and 4a (DeltaH = 138 +/- 3 kJ mol(-1) and DeltaS = 59 +/- 8 J mol(-1) K(-1)), are inconsistent with anomeric carboxylate assistance occurring during the hydrolysis reactions, and the likely cause for the enhanced reactivity of 5 in comparison to that of 4a is an increase in ground-state steric strain.     

电化学石英晶体阻抗系统研究金电极上Fe(CN)_6~(3-)/Fe(CN)_6~(4-)电化学过程

电化学过程的石英晶体阻抗分析法已用于现场获取电活性聚合物粘弹性等信息［１,２］．本文联用ＨＰ４３９５Ａ阻抗／网络／频谱分析仪和ＥＧ＆ＧＭ２８３恒电位仪开发出电化学石英晶体阻抗系统（ＥｌｅｃｔｒｏｃｈｅｍｉｃａｌＱｕａｒｔｚＣｒｙｓｔａｌＩｍｐｅｄａｎ...     

Letter: correlation between phosphorylation ratios by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis and enzyme kinetics

To identify the correlation between the phosphorylation ratios by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analysis and enzyme kinetics (K(m), V(max), and V(max)/K(m)) is important to understand whether MALDI-TOF MS can be applied for monitoring the properties of peptides that are substrates of protein kinases. The correlation between phosphorylation ratios and enzyme kinetics was examined using peptides for protein kinase C (PKC) and for 60 kDa phosphoprotein, encoded by the cellular sarcoma gene (c-Src). Phosphorylation ratios, analyzed by MALDI-ToF MS, showed higher correlation coefficient (r = > +0.7) for V(max)/K(m) compared with that (r = < -/+0.6) for K(m) or V(max). For ion modes, a higher correlation coefficient between phosphorylation ratios and V(max)/K(m) was identified in the positive mode (r = > +0.7) compared to that in the negative mode (r = < +0.5). These results suggest that MALDI-ToF MS is a useful tool to evaluate V(max)/K(m) of peptides for protein kinases.     

Characterization of adsorption of humic acid onto alumina using quartz crystal microbalance with dissipation

In this paper, a quartz crystal microbalance with dissipation monitoring (QCM-D) is used to investigate humic acid (HA) adsorption onto alumina (Al(2)O(3)). The amount of adsorption and layer structures of HA were determined by the real-time monitoring of resonance frequency and energy dissipation changes (Δf and ΔD). The effect of HA concentration, HA molecular characteristics (molecular weight and polarity), and pH on HA adsorption onto Al(2)O(3) were investigated. The mass of HA adsorption increases as the concentration of HA increases. The masses are about 24, 60, and 87 ng cm(-2) as the concentration of DOC is 1.0, 4.85, and 92.0 mg L(-1), respectively. The adsorbed layer of HA is more nonrigid, and the mass of HA adsorption is higher at weakly acidic pH values. It was 20, 80, 65, and 45 ng cm(-2) at pH values of 4.5, 5.5, 6.5, and 8.0, respectively. This reveals that efficient HA removal by coagulation at weakly acidic pH values is not just due to the hydrolysis of Al ions as previously presumed. The adsorbed layer of hydrophobic HA is more nonrigid than hydrophobic HA (fractionated by Amberlite XAD-8 resin), and the mass adsorption for the hydrophobic fraction is about four times higher than the hydrophilic fraction (120 ng cm(-2) and 30 ng cm(-2)). The method is of value in the research to establish a quantified calculation model for the coagulation process.     

Surface properties and enzymatic degradation of end-capped poly(l-lactide)

Surface properties and enzymatic degradation of poly(l-lactide) (PLLA) end-capped with hydrophobic dodecyl and dodecanoyl groups were investigated by means of advancing contact angle (θ_a) measurement, quartz crystal microbalance (QCM) and atomic force microscopy (AFM). The θ_a values of end-capped PLLA films were larger than those of non-end-capped PLLA films, suggesting that the hydrophobic dodecyl and dodecanoyl groups were segregated on the film surface. The weight changes of end-capped PLLA thin films during enzymatic degradation in the presence of proteinase K were monitored by using a QCM technique. The relatively fast weight loss of PLLA film occurred during first few hours of degradation, followed by a decrease in the erosion rate. The erosion rate of PLLA films at the initial stage of degradation was dependent on the chain-end structure of PLLA molecules, and the value decreased with an increase in the amount of hydrophobic functional groups. The surface morphologies of PLLA thin films before and after degradation were characterized by AFM. After the enzymatic degradation, the surface of non-end-capped PLLA films was blemished homogeneously. In contrast, the end-capped PLLA thin films were degraded heterogeneously by the enzyme, and many hollows were formed on the film surface. From these results, it has been concluded that the introduction of hydrophobic functional groups at the chain-ends of PLLA molecules depressed the erosion rate at the initial stage of enzymatic degradation.     

Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme

The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKa values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Br?nsted beta(lg) of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are (18)(V/K) bridge = 1.0170 and (15)(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Br?nsted beta(lg) of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the beta(lg) measured for monoester substrates, indicative of similar transition states. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.     

In Situ Acquisition of Equivalent Circuit Parameters of Crystal Resonance during Copper Deposition and Dissolution in Acidic Solution by Electrochemical Quartz Crystal Impedance System

Thequartzcrystalimpedancetechniqueisapowerfulmethodtocharacterizecrystalresonancesincemultipleinformationaboutcrystalresonanceincludingtheresonantfrequencyandchangesinsomephysicaland/orchemicalpropertiesofthetestsystemcanFigure1.Equivalentelectricalc...     

Characterization of the O(2)-evolving reaction catalyzed by [(terpy)(H2O)Mn(III)(O)2Mn(IV)(OH2)(terpy)](NO3)3 (terpy = 2,2':6,2"-terpyridine).

The complex [(terpy)(H(2)O)Mn(III)(O)(2)Mn(IV)(OH(2))(terpy)](NO(3))(3) (terpy = 2,2':6,2' '-terpyridine) (1)catalyzes O(2) evolution from either KHSO(5) (potassium oxone) or NaOCl. The reactions follow Michaelis-Menten kinetics where V(max) = 2420 +/- 490 mol O(2) (mol 1)(-1) hr(-1) and K(M) = 53 +/- 5 mM for oxone ([1] = 7.5 microM), and V(max) = 6.5 +/- 0.3 mol O(2) (mol 1)(-1) hr(-1) and K(M) = 39 +/- 4 mM for hypochlorite ([1] = 70 microM), with first-order kinetics observed in 1 for both oxidants. A mechanism is proposed having a preequilibrium between 1 and HSO(5-) or OCl(-), supported by the isolation and structural characterization of [(terpy)(SO(4))Mn(IV)(O)(2)Mn(IV)(O(4)S)(terpy)] (2). Isotope-labeling studies using H(2)(18)O and KHS(16)O(5) show that O(2) evolution proceeds via an intermediate that can exchange with water, where Raman spectroscopy has been used to confirm that the active oxygen of HSO(5-) is nonexchanging (t(1/2) > 1 h). The amount of label incorporated into O(2) is dependent on the relative concentrations of oxone and 1. (32)O(2):(34)O(2):(36)O(2) is 91.9 +/- 0.3:7.6 +/- 0.3:0.51 +/- 0.48, when [HSO(5-)] = 50 mM (0.5 mM 1), and 49 +/- 21:39 +/- 15:12 +/- 6 when [HSO(5-)] = 15 mM (0.75 mM 1). The rate-limiting step of O(2) evolution is proposed to be formation of a formally Mn(V)=O moiety which could then competitively react with either oxone or water/hydroxide to produce O(2). These results show that 1 serves as a functional model for photosynthetic water oxidation.     

Chiral capillary electrophoretic analysis of verapamil metabolism by cytochrome P450 3A4

Cytochrome P450 (CYP), which is one of the most important enzymes in human liver, is responsible for a large portion of the first-pass metabolism of drugs. Many studies have focused on the determination of CYP activity by substrate assays. Most of them used liquid chromatography (LC) as analytical technique, while only a few studies used capillary electrophoresis (CE) for the separation and quantitation of reaction components. In this study, the feasibility of using CE in an in vitro metabolism study with CYP was tested. Verapamil was chosen as the substrate for CYP 3A4 isozyme (Supersome). A chiral capillary electrophoretic method was developed and validated for the simultaneous determination of R,S-verapamil (VER) and their major metabolites, R,S-norverapamil (NOR). A method for CYP 3A4 activity assay was proposed with VER as a probe. At the same time, the enantioselective metabolism of VER was studied. Michaelis-Menten constants of R- and S-VER were determined. S-VER was metabolised faster and more extensively than R-VER, with K(m)=167+/-23 microM, V(max)=3,418+/-234 pmol/min/mg for S-VER, and K(m)=168+/-35 microM, V(max)=2,502+/-275 pmol/min/mg for R-VER.     

Study on degradation characteristics of chitosan by pepsin with piezoelectric quartz crystal impedance analysis technique.

Piezoelectric quartz crystal impedance analysis technique was applied to study the chitosanolytic activity of pepsin. The method is based on the viscosity-density reduction of chitosan solution during the enzymatic degradation process. Experiments examined the time courses of the variations of motional resistance (deltaR1) for a quartz crystal. By comparing the deltaR1 response curves under different degradation conditions, the effects of pH, temperature, enzyme and substrate concentration on the chitosanolytic activity of pepsin was investigated in detail. The results suggest that the optimum pH and temperature were 4.6 and 55 degrees C, respectively. Increasing aptly the enzyme or substrate concentration was in favor of the degradation of chitosan. Moreover, the influence of the degree of deacetylation (DD) on the enzymatic degradation was studied. The result indicates that chitosan with a lower DD was easier to be degrade compared with chitosan with a higher DD. Also, it was found that there was a good linear relationship between the deltaR1 response and the DD value. The regression equation was deltaR1 = 0.058 x DD-6.795 and the correlation coefficient was 0.987.     

Application of the NT solvent nucleophilicity scale to attack at phosphorus: solvolyses of N,N,N',N'-tetramethyldiamidophosphorochloridate

The specific rates of solvolysis of N,N,N',N'-tetramethyldiamidophosphorochloridate have been measured at 25.0 degrees C in 31 solvents. Analysis with the extended Grunwald-Winstein equation leads to sensitivities toward changes in solvent nucleophilicity (l) of 1.20 +/- 0.07 and toward changes in solvent ionizing power (m) of 0.69 +/- 0.04. The correlation is improved by omission of the four data points for 2,2,2-trifluoroethanol-ethanol mixtures (F-test value from 155 to 320) with very small reductions in both l and m values. Activation parameters are reported for eight of the solvolyses. The l and m values are very similar to those previously reported for solvolyses of several arenesulfonyl chlorides, consistent with a concerted substitution process. This assignment is supported by a large k(Cl)/k(F) ratio for hydrolysis and a corresponding ratio for hydroxide-assisted hydrolysis of 178. The stereochemistry of nucleophilic attack at tetracoordinate phosphorus(V) is discussed.     

Spectrophotometric determination of hydrogen peroxide by using the cleavage of Eriochrome black T in the presence of peroxidase

A new hydrogen donor for peroxidase, Eriochrome black T, was reported for the first time. Steady-state catalytic velocity depends upon enzyme and substrate concentrations, and a Michaelis-Menten K(m) value of 1.72x10(-5) mol l(-1) and a V(max) value of 4.43x10(-3) s(-1) were measured at pH 8.6. Trace amount of hydrogen peroxide (2x10(-7)-1.0x10(-5) mol l(-1)) was determined in aqueous solution by using the cleavage of Eriochrome black T catalyzed by peroxidase. The method is simple and practical, with high sensitivity and enzymatic activity.