Tonometric biosensor with a differential pressure sensor for chemo-mechanical measurement of glucose

A tonometric biosensor for glucose was constructed using a chemo-mechanical reaction unit and a differential pressure sensor. The reaction unit was fabricated by using both liquid and gas cells separated by an enzyme diaphragm membrane, in which glucose oxidase was immobilized onto the single (gas cell) side of the dialysis membrane. By applying glucose solution (0, 25.0, 50.0, 100, 150 and 200 mmol/l) into the liquid cell of the chemo-mechanical reaction unit, the pressure in the gas cell decreased continuously with a steady de-pressure slope because the oxygen consumption in the gas cell was induced by the glucose oxidase (GOD) enzyme reaction at the enzyme side of the porous diaphragm membrane. The steady de-pressure slope in the gas cell showed the linear relationship with the glucose concentration in the liquid cell between 25.0 and 200.0 mmol/l (correlation coefficient of 0.998). A substrate regeneration cycle coupling GOD with l-ascorbic acid (AsA: 0, 1.0, 3.0, 10.0 and 50.0 mmol/l; as reducing reagent system) was applied to the chemo-mechanical reaction unit in order to amplify the output signal of the tonometric biosensor. 3.0 mmol/l concentration of AsA could optimally amplify the sensor signal more than 2.5 times in comparison with that of non-AsA reagent.     

The use of a layering technique for enhancing stability of lyophilized reagents.

An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351. Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use. The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance. This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH. For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience. However, the working reagent required a pH of 10.0 upon reconstitution. Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution. This incompatibility was overcome by use of a layering technique. The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.     

Electron paramagnetic resonance identification of a highly reactive thiol group in the proximity of the catalytic site of human placenta glutathione transferase.

The reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.     

3-(N-Maleimido-propionyl)biocytin: a versatile thiol-specific biotinylating reagent

A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) biocytin (MPB), was synthesized and used to biotinylate various proteins via native or artificially induced sulfhydryl groups. In combination with appropriate avidin- or streptavidin-conjugated markers (i.e., fluorescent, enzyme-conjugated, electron-dense, etc.), MPB essentially constitutes a universal, multipurpose, thiol-specific probe. The reagent could be used to detect protein SH groups on dot blots with sensitivities in the femtomole range. The labeling was very specific for sulfhydryl groups or reduced S-S bonds; proteins lacking free SH groups were unlabeled by this method. Due to the long spacer between the biotinyl group and the reactive maleimide, improved adsorption of biotinylated proteins to avidin columns was achieved. An SH-containing enzyme (beta-galactosidase) was biotinylated with MPB, and the resultant biotinylated enzyme could be used as an efficient histochemical probe. The use of this reagent is recommended to biotinylate proteins which contain nonessential SH groups or which can be easily thiolylated prior to reaction with MPB.     

Synthesis of a reactive ATP analog and its preliminary application as an affinity labeling reagent

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP.PCP, was synthesized in an attempt to covalently label the binding sites for adenine nucleotides, especially ATP, of various enzymes which utilize adenine nucleotides as substrates, cofactors, inhibitors or allosteric effectors. This reagent rapidly inactivated rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD), myokinase (MK), and creatine kinase (CK) under very mild conditions. Adenine nucleotide substrates prevented the inactivation. In the case of GPD, complete inactivation was observed when 1 mol of the reagent per mol of enzyme subunit was incorporated into the enzyme. These results indicate that the present ATP analog may be useful as an affinity labeling reagent for various adenine nucleotide-dependent enzymes.     

Iodination of proteins by IPy2BF4, a new tool in protein chemistry

Iodination is a very useful method for protein characterization and labeling. However, derivatization chemistries used in most conventional iodination procedures may cause substantial alterations in protein structure and function. The IPy(2)BF(4) reagent [bis(pyridine)iodonium (I) tetrafluoroborate] has been shown to be an effective iodinating reagent for peptides. Herein we report the first application of IPy(2)BF(4) in protein iodination in an aqueous medium using three representative substrates: insulin, lysozyme, and the enzyme 1,3-1,4-beta-d-4-glucanohydrolase. Our results show that IPy(2)BF(4) has clear advantages over existing methods in that the reaction is quantitative, fast, and selective for the most accessible Tyr residues of a protein, and it preserves the functional integrity of the protein when moderate Tyr labeling levels are pursued.     

Optimization of a microarray sandwich-ELISA against hINF-gamma on a modified nitrocellulose membrane

The highly specific and highly sensitive ELISA (enzyme linked immunosorbent assay) technique is the most commonly used method for immunological diagnostics in general. In combination with protein microarrays and their ability to allow performing thousands of experiments in parallel, a promising tool for global analytical approaches with reduced consumption of time, analytes, and reagents is given. In this study a protein microarray-based sandwich-ELISA for human interferon-gamma (hINF-gamma) is established. In consideration of the immense importance of the surface chemistry, a new black nitrocellulose matrix that generates very high signal-to-noise ratios (SNR) and a very low autofluorescence was tested and optimized as microarray substrate. A validation of the applicability of the system was performed with a comparison to different commercially available systems. Experimental results show that the microarray-based ELISA is faster and easier to perform and shows a lower limit of detection (LOD) than a comparable system in a 96-well plate. The spotted slides with the capture antibody can be stored up to 1 month with no significant loss of signal intensity. A second model system with immobilized His-tagged restriction enzyme EcoRV and an anti-His antibody shows in coincidence the good applicability of the black nitrocellulose membrane and no cross-reactivity toward the ELISA.     

Ozone-3,6-dihydroxynaphtha-2,7-disulphonate chemiluminescence system is used for online ozone detection

The chemiluminescence (CL) reaction between ozone and 3,6-dihydroxynaphtha-2,7-disulphonate (DNDS) was found under alkaline conditions. Therefore, a novel CL system for ozone detection was established. The CL signal of the CL system is weak, and the CL signal is enhanced by adding nonionic surfactants. It was found that adding 16.4 g/l Triton X-100 can enhance the CL signal. The CL reagent activated by ultraviolet (UV) light produced a CL signal was nearly 10 times stronger than the CL reagent not activated by UV light; the CL signal was enhanced by adding 8 g/l NaHCO₃ to the CL reagent irradiated by UV light. Through the optimization of these test conditions, a high-selectivity, high-sensitivity online detection method for ozone CL was established. The linear range was 0.5–150 ppbv, and the limit of detection (LOD) was 0.092 ppbv (S/N = 3).     

Monoacylglycerol Acyltransferase-2 Is a Tetrameric Enzyme That Selectively Heterodimerizes with Diacylglycerol Acyltransferase-1

Acyl-CoA:monoacylglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze the two consecutive steps in the synthesis of triacylglycerol, a key process required for dietary fat absorption into the enterocytes of the small intestine. In this report, we investigated the tendency of MGAT2 to form an enzyme complex with DGAT1 and DGAT2 in intact cells. We demonstrated that in addition to the 38-kDa monomer of the MGAT2 enzyme predicted by its peptide sequence, a 76-kDa moiety was detected in SDS-PAGE without reducing agent and heat inactivation. The 76-kDa MGAT2 moiety was greatly enhanced by treatment with a cross-linking reagent in intact cells. Additionally, the cross-linking reagent dose-dependently yielded a band corresponding to the tetramer (152 kDa) in SDS-PAGE, suggesting that the MGAT2 enzyme primarily functions as a homotetrameric protein and as a tetrameric protein. Likewise, DGAT1 also forms a homodimer under nondenaturing conditions. When co-expressed in COS-7 cells, MGAT2 heterodimerized with DGAT1 without treatment with a cross-linking reagent. MGAT2 also co-eluted with DGAT1 on a gel filtration column, suggesting that the two enzymes form a complex in intact cells. In contrast, MGAT2 did not heterodimerize with DGAT2 when co-expressed in COS-7 cells, despite high sequence homology between the two enzymes. Furthermore, systematic deletion analysis demonstrates that N-terminal amino acids 35–80 of DGAT1, but not a signal peptide at the N terminus of MGAT2, is required for the heterodimerization. Finally, co-expression of MGAT2 with DGAT1 significantly increased lipogenesis in COS-7 cells, indicating the functional importance of the dimerization.     

Correcting a potential defect in an enzymatic cycle for NADP

An enzymatic cycle for NADP which uses as one of its enzymes glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has occasionally caused trouble due to failure to completely heat-kill this enzyme before the indicator step. It was found that a very small increase in pH was the cause of this. It was also found that the other two proteins present in the reagent greatly increase heat inactivation of the enzyme. The inactivation problem is completely overcome by keeping the pH below 7.2.     

The enzyme-mediated activation of radical source reaction: a new approach to identify partners of a given molecule in membrane microdomains

Important biological events associated with plasma membranes, such as signal transduction, cell adhesion, and protein trafficking, are mediated through the membrane microdomains. However, it is difficult to assess the issue of how they assemble under physiological conditions. We developed a new approach to identify partners of a given molecule on the cell surface in living cells. The important feature of this system, termed as enzyme-mediated activation of radical source, is that activation of cross-linking reagent arylazide-biotin tag can be accomplished not by ultraviolet light, but by an enzyme, horseradish peroxidase. By using this method, we found that many kinds of receptor tyrosine kinases are associated with β1 integrin whereas a few receptor tyrosine kinases are associated with ganglioside GM1 in HeLa S3 cells. This system is a comprehensive approach to identify interactions between cell surface molecules under living conditions. The advantages of this approach are as follows: (i) easy, high throughput, and without the need for special equipment, (ii) applicable to systematic approaches such as proteomic analysis, (iii) applicable to studies on the interactions among not only proteins but also glycans and lipids. The biochemical approach although the enzyme-mediated activation of radical source reaction will provide a new insight into a wide range of research concerning cis-interaction between biomolecules on the cell surface in living cells.     

The identification of a structurally important cysteine residue in the glycerol dehydrogenase from Bacillus stearothermophilus

Evidence is presented to demonstrate that the Zn2+ metallo-enzyme glycerol dehydrogenase from the thermophile Bacillus stearothermophilus has one cysteine residue per subunit which is only available for reaction with thiol reagents in the metal-depleted form of the enzyme. Modification of the metal-depleted enzyme by methyl methanethiosulphonate prevents the reactivation of the enzyme by Zn2+ ions and induces dissociation of the oligomer into subunits. The rate of reaction of the cysteine residue with the thiol reagent DTNB is limited by a factor other than reagent concentration and it is proposed that the reagent only reacts with the cysteine residue in dissociated monomers. The enzyme has been labelled at the single cysteine residue by radioactive iodo[2-3H]acetic acid. Two radiolabelled peptides have been isolated and sequenced; one peptide is a component of the other. Spectroscopic evidence suggests that the cysteine residue is not involved in ligation of the essential metal ion. Chemical modification studies using the reagent diethylpyrocarbonate have suggested that two histidines are involved in the ligation of the metal.     

Identification of a tyrosine residue responsible for <Emphasis Type="Italic">N</Emphasis>-acetylimidazole-induced increase of activity of ecto-nucleoside triphosphate diphosphohydrolase 3

Chemical modification in combination with site-directed mutagenesis was used to identify a tyrosine residue responsible for the increase in ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) nucleotidase activity after acetylation with a tyrosine-selective reagent, N-acetylimidazole. The NTPDase3 ATPase activity is increased more than the ADPase activity by this reagent. Several fairly well conserved tyrosine residues (252, 255, and 262) that are located in or very near apyrase conserved region 4a (ACR4a) were mutated. These mutants were all active, but mutation of tyrosine 252 to either alanine or phenylalanine eliminated the activity increase observed after N-acetylimidazole treatment of the wild-type enzyme. This suggests that the acetylation of tyrosine 252 is responsible for the increased activity. Stabilization of quaternary structure has resulted in increased enzyme activities for the NTPDases. However, mutation of these three tyrosine residues did not result in global changes of tertiary or quaternary structure, as measured by Cibacron blue binding, chemical cross linking, and native gel electrophoretic analysis. Nevertheless, disruption of the oligomeric structure with the detergent Triton X-100 abolished the increase in activity induced by this reagent. In addition, mutations that abolished the N-acetylimidazole effect also attenuated the increases of enzyme activity observed after lectin and chemical cross-linking treatments, which were previously attributed to stabilization of the quaternary structure. Thus, we speculate that the acetylation of tyrosine 252 might induce a subtle conformational change in NTPDase3, resulting in the observed increase in activity.     

Insertion of a disulfide-containing neurotoxin into E. coli alkaline phosphatase: the hybrid retains both biological activities.

We have inserted a disulfide-containing snake neurotoxin into the N-terminal end of Escherichia coli alkaline phosphatase, between residues +6 and +7 of the mature enzyme. For this purpose, we have designed a cloning and expression vector which allows insertion of foreign DNA between the corresponding codons, and visual selection of the desired recombinant clones upon recovery of phosphatase activity. The hybrid protein is exported to the bacterial periplasm, the alkaline phosphatase signal peptide is correctly processed, and both domains are functionally conformed. The phosphatase domain displays catalytic activity, and the inserted toxin is able to bind to its biological target, the nicotinic acetylcholine receptor. The hybrid molecule is remarkably stable and resistant to proteolysis. Crude periplasmic extract containing the hybrid can be used as a tracer-containing reagent in competitive enzymo-immuno and enzymo-receptor assays. We propose to use the system described in this paper for fast preparation of properly folded disulfide-containing enzymatic probes.     

Photoaffinity labelling of carnitine acetyltransferase with S-(p-azidophenacyl)thiocarnitine.

A photolabile reagent, p-azidophenacyl-DL-thiocarnitine, was synthesized and tested as a photoaffinity label for carnitine acetyltransferase (EC 2.3.1.7) from pigeon breast. p-Azidophenacyl-DL-thiocarnitine is an active-site-directed reagent for this acetyltransferase, since it is a competitive inhibitor (Ki 10 microM) versus carnitine. U.v. irradiation of a mixture of p-azidophenacyl-DL-thiocarnitine and enzyme produces irreversible inhibition. Acetyl-DL-carnitine protects the enzyme from inhibition by photoactivated p-azidophenacyl-DL-thiocarnitine. In the presence of 30 mM-2-mercaptoethanol as a scavenger, the relationship between loss of activity and photoincorporation of reagent suggests that one molecule of reagent is incorporated per molecule of inhibited enzyme. However, peptide maps of enzyme labelled with p-azidophenacyl[14C]thiocarnitine indicate that several (about six) tryptic peptides (of a possible 60-65) are modified. The presence of 5 mM-acetyl-DL-carnitine significantly decreases the incorporation of reagent in each labelled tryptic peptide.     

Studies by electron-paramagnetic-resonance spectroscopy and stopped-flow spectrophotometry on the mechanism of action of turkey liver xanthine dehydrogenase.

Studies by e.p.r. (electron-paramagnetic-resonance) spectroscopy and by stopped-flow spectrophotometry on turkey liver xanthine dehydrogenase revealed strong similarities to as well as important differences from the Veillonella alcalescens xanthine dehydrogenase and milk xanthine oxidase. The turkey enzyme is contaminated by up to three non-functional forms, giving molybdenum e.p.r. signals designated Resting I, Resting II and Slow. Slow and to a lesser extent Resting I signals are like those from the Veillonella enzyme, whereas Resting II is very like a resting signal described by K. V. Rajagopolan, P. Handler, G. Palmer & H. Beinert (1968) (J. Biol. Chem. 243, 3784-3796) for aldehyde oxidase. Another non-functional form that gives the Inhibited signal is produced on treatment of the enzyme with formaldehyde. Stopped-flow measurements at 450 nm show that, as for the milk enzyme, reduction by xanthine is rate-limiting in enzyme turnover. The active enzyme gives rise to Very Rapid and Rapid molybdenum(V) e.p.r. signals, as well as to an FADH signal. That these signals are almost indistinguishable from those of the milk enzyme, confirms the similarities between the active sites. There are two types of iron-sulphur centres that give signals like those in the milk enzyme, though with slightly different parameters. Quantitative reduction titration of the functional enzyme with xanthine revealed two important differences between the turkey and the milk enzymes. First, the turkey enzyme FADH/FADH2 system has a redox potential sufficiently low that xanthine is incapable of reducing the flavin completely. This finding presumably explains the very low oxidase activity. Secondly, whereas the Fe/S II chromophore in the milk enzyme has a relatively high redox potential, for the turkey enzyme the value of this potential is lower and similar to that of its Fe/S I chromophore.     

On site heterogeneity in sturgeon muscle GPDH: a kinetic approach

The kinetics and stoichiometry of the reaction of sturgeon muscle glyceraldchyde-3-PO₄-dehydrogenase (GPDH) with the disulfide interchange reagent bis(2,2' dithio-bis(5-nitrobenzoate) (DTNB) has been studied in detail. The native enzyme, a tetramer of covalently identical subunits, reacts relatively rapidly with precisely four equivalents of reagent, although there are three cysteine residues per subunit (12 per tetratner). Reaction of these four cystcines leads to total catalytic inactivation; the extent of inactivation is proportional to the fractional reaction. The rate of reaction is dependent on the extent of bound NAD: reactivity being very much greater at unliganded sites. The reaction with apo-enzyme is fastest, bimolecular and monophasic. Over a wide range of NAD concentration, however, the reaction of enzyme with a large molar excess of reagent is precisely biphasic, and each individual kinetic experiment can be analytically described by two pseudo first-order (NAD concentration-dependent) rate constants and two unequal NAD concentration-insensitive amplitudes. The biphasicity in rate is quantitatively explainable on the basis of a C₂ symmetry for the tetrameric subunits with a tighter binding of NAD at two of the four sites, if high reactivity is exclusively dependent on the absence of bound NAD. The inequality in the two amplitudes, however, requires either a more complex or a more dynamic model. Arguments are presented for the appropriateness of a C₂ symmetry model in which intramolecular transconformational isomerization of tight and loose NAD binding sites is possible. The equilibrium constant for the isomerization is estimable from the macroscopic specific rates and amplitudes. This “flip-over” C₂ symmetry model is apropos to all situations of negative cooperativity in ligand binding to tetramers, as is discussed.     

Affinity labelling of alcohol dehydrogenases. Chemical modification of the horse liver and the yeast enzymes with alpha-bromo-beta(5-imidazolyl)-propionic acid and 1,3-dibromoacetone.

1. DL-alpha-Bromo-beta(5-imidazolyl)-propionic acid is a potential affinity labelling reagent for metallo-enzymes. It has been used with the alcohol dehydrogenases from liver and yeast. The liver enzyme is chemically modified and inactivated in a Michaelis-Menten-type reaction, where one molecule of the reagent is bound per subunit. The enzyme is protected from the inhibitor in a competitive manner by imidazole, 2,2'-dipyridyl, 1,10-phenanthroline and cyclohexanone, which all combine with the active-site zinc. The protection by chloride, acetate and NADH, which are considered to bind at the general anion binding site, is not strictly competitive. Inactivation has an optimum at pH 8.5. For the liver enzyme, the reagent was found to decrease the initial rate of ethanol oxidation. Prior to the irreversible alkylation of Cys-46, reversible binding is shown to occur at the active-site zinc atom. The yeast enzyme was extremely resistant to the reagent and no specific modification was found. 2. The potential affinity labelling and crosslinking reagent, symmetrical 1,3-dibromoacetone although unstable, has also been used for chemical modification. With the liver enzyme, concentrations below 5 mM gave a reaction of the Michaelis-Menten-type at pH 7.0. Several ligands known to complex with the active-site region protect the enzyme against the reagent. Dibromoacetone gave rapid inactivation of the yeast enzyme. Despite the fact that a pseudo-first-order reaction was observed with respect to enzyme as well as inhibitor, no saturating effect was found. In this work, dibromoacetone reacted like a monofunctional reagent.     

Immunochromatographic membrane strip assay system for a single-class plasma lipoprotein cholesterol, exemplified by high-density lipoprotein cholesterol measurement

In assessing risk factors of coronary heart disease, a membrane immunochromatographic system that minimizes requirements of instrument and reagent handling was investigated by utilizing high-density lipoprotein (HDL) cholesterol (HDL-C) as model analyte. The system is composed of four functional membrane strip pads connected in sequence as follows (from the bottom): immunoseparation based on the biotin-streptavidin reaction; catalytic conversion of cholesterol to hydrogen peroxide; production of a colorimetric signal; and induction of a continuous wicking of medium. For immunochromatography, a monoclonal antibody, specific to apolipoprotein B100 that is present on the surfaces of low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL), with a high binding constant (5 x 10(10) L/mol), was raised and chemically conjugated to streptavidin. The conjugate was first reacted with lipoprotein particles, and this mixture was absorbed by the capillary action into the biotin pad of the system. After being transferred by medium, immunocapture of LDL and VLDL particles onto the biotin pad took place, and in situ generation of a colorimetric signal in proportion to HDL-C occurred consecutively. The capture was selective as well as effective (minimum 88% of LDL and VLDL in clinical concentration ranges), and the detection limit of the HDL-C was far lower than 20 mg per 100 mL. The same concept may also be applicable to LDL cholesterol measurement provided suitable antibodies specific to HDL and VLDL are available.     

Affinity labelling of 5-aminolevulinic acid dehydratase with 2-bromo-3-(5-imidazolyl)propionic acid

2-Bromo-3-(5-imidazolyl)propionic acid, a zinc-directed thiol reagent, inactivates the enzyme 5-aminolevulinic acid dehydratase from bovine liver (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing, EC 4.2.1.24). The substrate, 5-aminolevulinic acid, completely protects against inactivation. The reagent inhibits the zinc-containing enzyme to a greater extent than the zinc-deprived enzyme; and it competes with the zinc chelator 1,10-phenanthroline. The reagent alkylates essential sulfhydryl groups of the enzyme, since the extent of the inactivation depends on the reduction of the enzyme protein by thiol compounds. It is concluded that the zinc site, the substrate site and the essential sulfhydryl groups are in close proximity in the active site.