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PubMed2002

NS3 protease of Langat tick-borne flavivirus cleaves serine protease substrates.

Langat (LGT) virus, initially isolated in 1956 from ticks in Malaysia, is a naturally occurring nonpathogenic virus with a very close antigenicity to the highly pathogenic tick-borne encephalitis (TBE) Western subtype virus and TBE Far Eastern subtype virus. NS3, the second largest viral protein of LGT virus, is highly conserved among flaviviruses and contains a characteristic protease moiety (NS3 pro). NS3 pro represents an attractive target for anti-protease molecules against TBE virus. We report herein a purification method specially designed for NS3 pro of LGT using a strategy for proper refolding coupled with the enzymatic characterisation of the protein. Different p-nitroanilide substrates, defined on canonic sequences for their susceptibility to Ser-protease, were applied to the proteolytic assays of the protein. The highest values were obtained from substrates containing an Arg or Lys (amino acid) residue at the P1 position. This purification method will facilitate the future development of reliable testing procedures for anti-proteases directed to NS3 proteins.

Amino Acid Sequence↗

PubMed1995

BDM compared with P(i) and low Ca2+ in the cross-bridge reaction initiated by flash photolysis of caged ATP.

Using flash photolysis of caged ATP in skinned muscle fibers from rat psoas, we examined the inhibitory effects of 2,3-butanedione monoxime (BDM) on the contraction kinetics and the rate of ATP hydrolysis of the cross-bridges at approximately 10 degrees C. The hydrolysis rate was estimated from the stiffness records. The effects of BDM were compared with those of orthophosphate (P(i)) and of reduction in [Ca2+] (low Ca2+), and it was found that i) BDM and low Ca2+ inhibited ATPase activity to the same extent as they inhibited the steady tension, whereas P(i) inhibited ATPase activity much less than tension; ii) BDM and P(i) decreased tension per stiffness during the steady contraction more than did low Ca2+; iii) neither BDM nor low Ca2+ affected the initial relaxation of the fiber on release of ATP, but P(i) slightly slowed it; and iv) BDM hardly influenced the rate of contraction development after relaxation, although P(i) and low Ca2+ accelerated it. We concluded that BDM inhibits the Ca(2+)-regulated attachment of the cross-bridges and force-generation of the attached cross-bridges.

Adenosine Triphosphate↗

PubMed1998

Depolarized resonance light scattering by porphyrin and chlorophyll a aggregates.

A quantum mechanical model is developed for the observed resonance enhancement of light scattering by aggregates of electronically interacting chromophores. Aggregate size, monomer oscillator strength, extent of electronic coupling, and aggregate geometry are all important determinants of intensity in resonance light scattering (RLS) spectra. The theory also predicts the value of the depolarization ratio (rho(v)(90)) of RLS for a given aggregate geometry. These results are used to interpret the RLS depolarization ratios of four aggregates: tetrakis(4-sulfonatophenyl)porphine aggregated at low pH (rho(v)(90) = 0.17 at 488 nm), trans-bis(N-methylpyridinium-4-yl)-diphenylporphinato copper(II) aggregated in 0.2 M NaCl solution (rho(v)(90) = 0.13 at 450 nm) and on calf thymus DNA (rho(v)(90) = 0.20 at 454 nm), and chlorophyll a aggregates in formamide/water (rho(v)(90) = 0.23 and 0.32 at 469 and 699 nm, respectively). The analysis is consistent with a J-aggregate geometry for all four systems. Furthermore, the specific values of rho(v)(90) allow us to estimate the orientation of the monomer transition dipoles with respect to the long axis of the aggregate. We conclude that depolarized resonance light scattering spectroscopy is a powerful probe of the geometric and electronic structures of extended aggregates of strong chromophores.

Animals↗

PubMed2000

Suitability and limitations of methods for characterisation of activity of malto-oligosaccharide-forming amylases.

The suitability and limitations of essential methods and reference substrates used for characterisation of activity of amylolytic enzymes is investigated. Saccharogenic, chromogenic and chromatographic methods are included. The results are discussed in relation to the measurement of reaction rates, determination of action mode and product specificity and the impact on identification and nomenclature of malto-oligosaccharide-forming amylases. An accurate determination of reaction rates using the saccharogenic methods strongly depends on the degree of polymerisation (DP) of the standards used and the hydrolysis products formed by the amylase. Particularly the use of glucose as standard can lead to overestimates due to the differences in the reducing potential of glucose and malto-oligosaccharides. The reliability of the chromogenic methods for determination of action mode depends on the DP of the substrate and the specificity of the amylase. For a characterisation of the starch hydrolysis products and the variation in the DP during hydrolysis, high performance anion-exchange chromatography with pulsed amperometric detection provided a fast and reliable method. A literature survey revealed varying and inconsistent use of nomenclature of malto-oligosaccharide forming amylases. Therefore a systematic approach identifying three main classes of activity is suggested using not only the mode of action and the DP of the major product but also the stage of hydrolysis at which this product is formed.

Amylases↗

PubMed2003

Enzymatic synthesis of beta-xylanase substrates: transglycosylation reactions of the beta-xylosidase from Aspergillus sp.

A beta-D-xylosidase with molecular mass of 250+/-5 kDa consisting of two identical subunits was purified to homogeneity from a cultural filtrate of Aspergillus sp. The enzyme manifested high transglycosylation activity in transxylosylation with p-nitrophenyl beta-D-xylopyranoside (PNP-X) as substrate, resulting in regio- and stereoselective synthesis of p-nitrophenyl (PNP) beta-(1-->4)-D-xylooligosaccharides with dp 2-7. All transfer products were isolated from the reaction mixtures by HPLC and their structures established by electrospray mass spectrometry and 1H and 13C NMR spectroscopy. The glycosides synthesised, beta-Xyl-1-->(4-beta-Xyl-1-->)(n)4-beta-Xyl-OC6H4NO2-p (n=1-5), were tested as chromogenic substrates for family 10 beta-xylanase from Aspergillus orizae (XynA) and family 11 beta-xylanase I from Trichoderma reesei (XynT) by reversed-phase HPLC and UV-spectroscopy techniques. The action pattern of XynA against the foregoing PNP beta-(1-->4)-D-xylooligosaccharides differed from that of XynT in that the latter released PNP mainly from short PNP xylosides (dp 2-3) while the former liberated PNP from the entire set of substrates synthesised.

Aspergillus↗

PubMed1998

Synthesis of aryl 3'-sulfo-beta-lactosides as fluorogenic and chromogenic substrates for ceramide glycanases.

We have previously reported that 4-methylumbelliferyl 6'-O-benzyl-beta-lactoside (2) is a useful substrate for a fluorometric assay of ceramide glycanase (CGase) (L.-X. Wang, N. V. Pavlova, S.-C. Li, Y.-T. Li and Y. C. Lee, Glycoconjugate J., 13 (1996) 359-365). The introduction of a 6-O-benzyl group at the terminal Gal efficiently protected the substrate from its hydrolysis by exo-galactosidase, permitting the assay of CGase in crude biological materials. However, a drawback of this substrate is its low water-solubility and relatively high Km (at a mM level). Introduction of a sulfate group into 4-methylumbelliferyl beta-lactoside (1) led to the formation of 4-methylumbelliferyl 3'-O-sulfo-beta-lactoside (3), which was found to be a more effective substrate than 2. Moreover, the presence of a 3'-O-sulfate group not only increases the water solubility tremendously, but also protects the substrate from cleavage by exo-beta-galactosidase as the 6'-O-benzyl group in 2 does. In addition to the fluorogenic substrate (3), two sulfated chromogenic substrates, N-tetradecanoyl-4-O(3'-sulfo-beta-lactosyl)-3-nitro-L-tyrosine methyl ester (9) and 2-N-(tetradecanoylamino)-4-nitro-phenyl 3'-sulfo-beta-lactoside (12), were synthesized and their suitability for a photometric assay of CGase was evaluated. Substrates 9 and 12, with a long fatty acid chain attached to the aglycon part, have a Km value close to that of the natural substrate GM1 (at a microM level).

Chromogenic Compounds↗

PubMed1997

Inhibition of human leukocyte elastase activity by chondroitin sulfates.

Chondroitin sulfates with different properties and devoid of appreciable anticoagulant activity were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Fractions with various mass and the same charge density were tested. The chondroitin sulfates with a molecular mass greater than about 2000 inhibited human leukocyte elastase activity to the same extent, whilst the fractions with a molecular mass of 1960 and 1020 were much less effective. The percentage inhibition of human leukocyte elastase activity increased based on the charge density of chondroitin sulfates. In particular, the inhibition of the enzymatic activity decreased with the percentage of non-sulfated disaccharide and increased with the amount of disaccharide-2,6-disulfated.

Animals↗

PubMed2000

Direct colorimetric method for determination of organophosphates in human urine.

A simple and sensitive method for determination of organophosphorus pesticides in human urine was developed by detecting the color complexes which resulted from reactions of organophosphorus pesticides and 4-(4-nitrobenzyl)pyridine (NBP) in urine. Based on studies of reaction conditions, e.g. reaction temperature and time, and reagent concentration, a colorimetric method was established. A 0.1-ml volume of NBP (45% in acetone) was added to a 1.0-ml volume of a urine sample, and the mixture was heated at 100 degrees C for 20 min. After cooling, 0.1 ml of tetraethylenepentamine was added. The organophosphorus pesticides showed a characteristic purplish blue color and the coloring complexes which were produced were stable for several hours. Furthermore, these complexes could be determined spectrophotometrically. The detection limits were 0.10-10 microg/ml in urine. The required time for analysis was approximately 30 min for one sample. Comparing the result of the proposed method with those of the GC-MS method, the results were similar for the 12 poisoning cases studied. Thus, the proposed method is useful for detection of these pesticides in critical care practices.

Chelating Agents↗

PubMed1984

Serum iron determination using ferene triazine.

The synthesis and characterisation of a new colorimetric reagent for iron, 3-(2-pyridyl)-5,6-bis(2-furyl)-1,2,4-triazine are described. Peak assignments and chemical shifts of the high resolution 13C nmr spectrum are given. The reagent forms a tris iron(II) complex with epsilon max of 32000 Lcm-1 mol-1 at 577 nm. The results of precision, linearity and recovery studies for use of the reagent in direct serum iron determinations are reported. Thiourea effectively suppresses copper interference for the serum iron determinations. With the procedure used, the presence of hemolysed cells in the serum had no effect on serum iron levels recorded.

Chromogenic Compounds↗

PubMed1989

A new colorimetric assay for purine nucleoside phosphorylase.

A new colorimetric procedure for the determination of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) activity is described. In this procedure, the hydrogen peroxide formed in the PNP-xanthine oxidase reaction is used to oxidize the chromogenic reagents--3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone using the enzyme peroxidase. The rate of enzyme reaction is followed at 520 nm. This procedure correlated well with the UV method (290 nm), with a correlation coefficient of 0.98 (P less than 0.005). Within-run and between-run precision (CV) were less than 2.8% and 3.7%, respectively. Here we also describe an optimized NAD-dependent method (340 nm) for PNP determination. The colorimetric method is superior to both the 340 nm and the UV methods in terms of both sensitivity and precision. The mean erythrocyte PNP activity for 17 healthy subjects was 11.88 U/mL packed cells for the NAD-dependent method and 13.22 U/mL packed cells for the colorimetric method.

Chromogenic Compounds↗

PubMed2000

Nonenzymatic elimination of ascorbic acid in clinical samples.

OBJECTIVES: Ascorbic acid interferes significantly in the oxidative reaction of chromogenic reagents by peroxidase and hydrogen peroxide. Currently, ascorbate oxidase is commonly utilized for eliminating the interference of ascorbic acid in the oxidative colorimetric reaction. This enzyme, however, displays several disadvantages, such as high cost, variation from lot to lot, and low stability. We applied a series of commercially available and stable radicals (ascorbic acid quenchers [AAQs]) for nonenzymatic quenching of ascorbic acid in the uricase-based uric acid determination in serum and urine. DESIGN AND METHODS: In order to evaluate the quenching activity of AAQs, a commercially available uric acid detection kit was used. TBA-80FR.NEO biochemical analyzer was utilized for the assay. RESULTS: 4-Hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy free radical (AAQ-2) was the most effective ascorbic acid quencher among the four stable radicals, and the uric acid assay suffered no interference by AAQ-2. The ascorbic acid quenching ability of 2 mmol/L of AAQ-2 in reagent solution (reagent-I) was > or = 2 U/ml ascorbate oxidase in reagent solution. CONCLUSIONS: AAQ-2 was proven to be a suitable quencher of ascorbic acid in clinical samples.

Ascorbate Oxidase↗

PubMed1999

Reactive oxygen intermediates and reactive nitrogen intermediates in copper intrauterine device users.

The mechanism of action of copper in copper intrauterine devices (Cu IUD) as an antimicrobial agent is not well understood. Copper and iron are supposed to be responsible for release of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), which are very active in the presence of infection. The copper in a copper IUD could be responsible for limiting pelvic inflammatory disease. The present study was composed of 20 IUD seekers in whom ROI and RNI were studied before insertion of Cu IUD and then at 1, 4, and 12 weeks afterward. ROI showed a rise after insertion, whereas RNI showed a steady decline. Hence, it is presumed that the rise in ROI could be responsible for both the bactericidal effect of Cu IUD and also for the fall in RNI.

Chromogenic Compounds↗

PubMed1997

Localization and activity of tissue factor in human aortic atherosclerotic lesions.

Tissue factor (TF) is a transmembrane protein that serves as the major initiator of the blood coagulation cascade. The overexpression of TF antigen and mRNA has previously been reported in advanced atherosclerotic lesions. Recently TF procoagulant activity has also been identified in these lesions. However, localization and activity of TF in various stages of atherosclerosis have not yet been reported. We studied TF localization and its activity in three stages of the human atherosclerotic lesions (diffuse intimal thickening, fatty streak, and atheromatous plaque). The thoracic aortas were obtained from 23 autopsy cases and were examined immunohistochemically using an anti-human TF polyclonal antibody and biotinylated factor VIIa (FVIIa) as a probe to test the FVIIa-binding ability of TF. In addition, the TF-mediated activation of factor X (FX) was quantitatively assessed using a chromogenic assay. In lesions of the diffuse intimal thickening and the fatty streak, almost all of intimal smooth muscle cells (SMCs), macrophages, and endothelial cells were positive for TF. In the atheromatous plaques, TF antigen was detected extensively in the extracellular matrix as well as in the intimal cells. TF in all stages of atherosclerotic lesions had the ability to bind biotinylated FVIIa. TF activity was detected in each lesion and was more prominent in fatty streaks and atheromatous plaques than in the diffuse intimal thickening. These results indicate that active TF is expressed in the early stage of atherosclerotic lesions as well as in the advanced stage, and it contributes to the thrombotic property of human atherosclerotic lesions.

Adolescent↗

PubMed2002

Affinity panning of peptide libraries using anti-streptokinase monoclonal antibodies: selection of an inhibitor of plasmin(ogen) active site.

To select sequences complementary to their binding sites, two anti-streptokinase (SK) monoclonal antibodies (mAbs), A4.5 and A5.5, were used in biopanning of 15-mer and hexamer phage-displayed peptide libraries, respectively. mAb A4.5 inhibits the catalytic activity of streptokinase-plasminogen activator complex (SKPAC), the binding of plasminogen to SK and the binding of human anti-SK polyclonal Abs to SK. All clones selected from the 15-mer peptide library by mAb A4.5 had identical nucleotide and amino acid sequences, RSVYRCSPFVGCWFG. An 11-mer peptide (peptide A4.5, YRCSPFVGCWF) derived from this sequence inhibited the binding of mAb A4.5 and human anti-SK polyclonal Abs to SK as well as the catalytic activity of both SKPAC and plasmin. The binding of the second mAb (mAb A5.5) to SK is lost upon interaction of SK with plasminogen, suggesting that sequences selected by this mAb are likely associated with the C-terminal cleavage site of SK. Biopanning of a hexamer peptide library with mAb A5.5 selected the sequence RYLQDY that is homologous to residues 324-328, adjacent to one possible C-terminal cleavage site in SK. A 10-mer synthetic peptide (LDFRDLYDPR) corresponding to residues 321-330 in SK specifically inhibited the binding of mAb A5.5 to SK. The selection and characterization of these two peptides enhances our understanding of SK structure, maps an antigenic epitope, and identifies a peptide inhibitor of plasminogen activation.

Amino Acid Sequence↗

PubMed1983

Enzyme-linked immunosorbent assay for anti-DNA antibodies using fluorogenic and colorigenic substrates.

An enzyme-linked immunosorbent assay is described for the assay of anti-DNA antibodies. The method employs plastic surface for immobilization of the antigen and alkaline phosphatase-linked rabbit anti-human IgG for the detection of immune complex using PNP-P and 4MU-P as substrates. The sensitivity of the assay increased by as much as 16-fold when fluorogenic substrate was used instead of conventional PNP-P and could therefore be employed for the detection of low avidity antibodies. Using PNP-P as substrate 57% of SLE patients were positive for DNA antibody, but if 4MU-P was introduced as substrate, 71% gave a positive response. Moreover, using a fluorogenic substrate, it was possible to minimise the amount of antigen (2 nM bp). The technique is simple, reproducible and of high sensitivity.

Alkaline Phosphatase↗

PubMed1997

A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.

This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H2O2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550). Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H2O2 oxidation. Using 50 microM A6550, the change in fluorescence due to H2O2 generated from phorbol 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5 x 10(4) and 5 x 10(4) cells, respectively. The increase in fluorescence from A6550 is specifically due to H2O2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H2O2 dose response is linear as long as the ratio of A6550:H2O2 in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H2O2. When the concentrations of A6550 and HRP were 10 microM and 0.2 U/ml, respectively, as low as 2 pmol of H2O2 could be reliably measured. The sensitivity of A6550/H2O2 assay is found to be at least 10-fold higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H2O2 release from activated granulocytes can be easily adapted to other cell types which generate H2O2.

Catalase↗

PubMed2000

A stable and highly sensitive 3,3',5,5'-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays.

3,3',5,5'-Tetramethylbenzidine (TMB) is a widely used chromogen for horseradish peroxidase-based detection systems because it yields reaction products with high absorption coefficients and lacks carcinogenicity. Unfortunately, TMB is labile and poorly soluble in aqueous buffers and such solutions must be freshly prepared before each experiment. Moreover, substrate depletion can occur under assay conditions. To overcome these problems we have developed a two-component TMB substrate system which has a lower detection limit and is more sensitive than many commercially available TMB reagents when compared in microtiter plate enzyme-linked immunosorbent assays. Both components of the substrate system are stable for at least 1 year at 4 degrees C and the usual discoloration of TMB stock solutions is prevented by the addition of a stabilizer that decomposes upon mixing of the two components.

Animals↗

PubMed2001

Measurement of factor VIII activity of B-domain deleted recombinant factor VIII.

The factor VIII activity of B-domain deleted recombinant factor VIII (BDDrFVIII) measured by activated partial thromboplastin time (APTT)-based one-stage assays is approximately 50% of the activity obtained by the chromogenic assay. Similar results have been reported for the two licensed full-length recombinant factor VIII products. In view of these findings, comprehensive studies have been undertaken to find the cause of the assay differences. Only the phospholipid reagent, used as a platelet substitute in the one-stage assay, proved to be crucial for explaining the assay difference. When platelet-rich plasma was used as the source of phospholipid in the one-stage assay, the factor VIII activity assay results correlated well with those measured by the chromogenic assay. Similar results were obtained when the platelets were replaced by liposomes prepared using platelet factor 3 (PF3) as a model that has a low content (5% to 10%) of phosphatidylserine (PS). In contrast, the use of liposomes with 20% to 30% PS, as in the crude lipid extracts used in ordinary APTT reagents, resulted in underestimation of the factor VIII activity. Antigen measurements using an enzyme-linked immunosorbent assay (ELISA) method demonstrated a good correlation between the antigen and chromogenic activity, but not always between antigen and one-stage activity results. Based on these findings and the clinical data, it can be concluded that the chromogenic assay most accurately measures the functional activity of BDDrFVIII. However, modifications of the one-stage assay, such as the use of a product-specific standard or development of a PF3-like phospholipid reagent, could address the observed assay discrepancies.

Chromogenic Compounds↗