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At least 127 records · Page 7Linked to original sources
PubMed1997

Configuration of heptopyranoside and heptofuranoside side chains: 2-anthroate, a powerful chromophore for exciton coupled CD.

The absolute configuration of the acyclic side chain of heptopyranosides and heptofuranosides was determined by exciton coupled CD, employing the strongly fluorescent 2-anthroate chromophore. The usage of this chromophore offers significant improvements over previous chemical and spectroscopic procedures since its intense fluorescence greatly facilitates the isolation and HPLC purification at the nanogram scale. The large amplitudes of the bisignate spectra allow CD manipulations in the 1 x 10(-7) M range.

Anthracenes↗
PubMed1992

Demonstration of proteases in basal cell carcinomas. A histochemical study using amino acid-4-methoxy-2-naphthylamides as chromogenic substrates.

BACKGROUND: Proteases are reported to play an essential part in the proliferative, invasive, and metastasizing behavior of malignant tumors. The aim of the current study was to determine the activity and localization of proteases in basal cell carcinomas (BCC) histochemically. METHODS: Various proteases were identified histochemically in frozen sections of BCC. The following amino acid-4-methoxy-2-naphthylamides (MNA) were used as chromogenic substrates:alanine-MNA for the detection of aminopeptidase M (APM), glycyl-proline-MNA for dipeptidyl peptidase IV (DPP IV), lysyl-proline-MNA and lysyl-alanine-MNA for dipeptidyl peptidase II (DPP II), glycyl-arginine-MNA for dipeptidyl peptidase I (DPP I), and carbobenzoxy (CBZ)-arginyl-arginine-MNA for cathepsin B. RESULTS: APM activity was high in the peritumorous connective tissue, whereas the tumor epithelium and epidermis had negative results. DPP IV showed a highly positive reaction in both tumor epithelium and surrounding connective tissue. Cathepsin B and DPP I reacted strongly in the tumor epithelium but not in the peritumorous connective tissue. CONCLUSIONS: The marked activity of APM, DPP IV, DPP I, and cathepsin B may be related to the proliferation and invasive growth of BCC. The distribution of the activity of APM and DPP IV indicates dynamic interactions between the tumor epithelium and the adjacent connective tissue in the neoplastic process.

2-Naphthylamine↗
PubMed2000

Plasma beta-N-acetylhexosaminidase isoenzyme composition and temperature conversion factors.

Using the chromogenic substrate 3,3'-dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide for the activity determination of plasma beta-N-acetylhexosaminidase (Hex), the temperature conversion factors (TCF) offer a highly significant positive correlation with the relative proportion of Hex B isoenzyme (P< 0.001). The calculation of TCF 37 degrees/25 degrees C allows the isoenzyme composition of Hex to be determined quickly and cheaply. The results may be superimposed over those obtained in a previously described method based on the calculation of the enzyme's activation energy using four temperatures. However, the use of TCF 37 degrees/30 degrees C does not appear to comply with the required demands.

Autoanalysis↗
PubMed2002

Enzyme fingerprints of activity, and stereo- and enantioselectivity from fluorogenic and chromogenic substrate arrays.

A series of stereochemically and structurally diverse fluorogenic and chromogenic substrates for hydrolytic enzymes has been synthesized and used to characterize enzyme activity profiles of esterases, lipases, proteases, peptidases, phosphatases, and epoxide hydrolases. The substrates used are particularly resilient to nonspecific reactions due to their mechanism of activation. The activities recorded with the individual substrates are therefore remarkably reproducible, and enable us to use the overall pattern of activity as a specific fingerprint for the enzyme sample. Fingerprints of activity, and enantio- and stereoselectivity are displayed as arrays of color-scale squares that are easily analyzed visually. Such fingerprints might be useful for quality control, enzyme discovery, and possibly for addressing the issue of functional convergence in enzymes.

Animals↗
PubMed2000

A highly sensitive method for the analysis of nitrite ions by capillary zone electrophoresis using water-soluble aminophenylporphyrin derivative as chromogenic reagent.

The water soluble 5-p-aminophenyl)-10,15,20-tris(p-sulfonatophenyl) porphyrin, 4, acts as an extremely efficient chromogenic reagent for the detection of very low amounts of nitrites. The amino group of porphyrin 4 reacts smoothly with nitrite in acidic conditions 0.2 M HCl) producing the corresponding diazo-porphyrin derivative which is stable and does not show any appreciable hydrolysis to phenol within 6 h. The reaction is carried out in the presence of 25 mM heptakis-(2,6-di-O-methyl)-beta-cyclodextrin that prevents precipitation of the protonated form of porphyrins 4 or 5 due to the formation of strong inclusion complexes. The capillary zone electrophoresis of the diazoporphyrin and amino-porphyrin mixture shows severe peak tailing. However, symmetrical peaks can be obtained by adding 5 mM beta-cyclodextrin to the background electrolyte (20 mM phosphate buffer, pH 7.0). Calibration curve for nitrite analysis is linear up to 0.25 mM nitrite and the detection limit (at a signal-to-noise ratio of 3) has been estimated to be a 1 microM (50 ppb) of nitrite concentration in aqueous solutions.

Chromogenic Compounds↗
PubMed2001

A fluorogenic assay using pressure-driven flow on a microchip.

A fluorogenic assay for human T-cell phosphatase (TCPTP) was conducted on an etched glass microchip using pressure driven flow. The TCPTP enzyme catalyzes the removal of a phosphate group from 6,8-difluoro-4-methylumbelliferyl/phosphate (DiFMUP) to produce the fluorogenic product 6,8-difluoro-4-methylumbelliferone (DiFMU). Enzyme assays with real-time on-chip dilution were performed in both low-viscosity (1 cP) buffer and an enzyme solution containing 50% glycerol (6 cP). Single side channels connect a series of reagent wells to a main channel where the fluorescent product of the enzyme reaction passes the detector region. Flow regulation of mixed viscosity fluids requires a pressure control on each arm of the chip contributing to the overall flow. An 8-channel pressure controller was built to regulate the air pressure above all wells feeding channels of the chip, thereby controlling the dilution ratios of buffer, substrate and enzyme. Well pressures maintained a constant concentration of enzyme in the detector channel while adjusting the flow contribution of substrate and buffer. The substrate concentration was stepped over two orders of magnitude while verifying fluid dilutions using marker dyes. The kinetic parameters, Km, Vmax, and Kcat, showed good agreement with the values determined using a standard well plate and fluorometer.

Chromogenic Compounds↗
PubMed1999

Beta2-glycoprotein I is necessary to inhibit protein C activity by monoclonal anticardiolipin antibodies.

OBJECTIVE: To clarify mechanisms of the thrombosis associated with anticardiolipin antibodies (aCL), we examined the effects on activated protein C (APC) of monoclonal aCL and beta2-glycoprotein I (beta2GPI), which is required for formation of the epitopes of aCL. METHODS: We developed the chromogenic assay, in which the degradation of coagulation factor Va by APC is reflected in the reduced generation of thrombin from prothrombin, using soybean trypsin inhibitor to inhibit APC. APC activities were measured in the presence and absence of 3.4 microM beta2GPI and/or 2.5 microg/ml of IgM monoclonal aCL (EY2C9 and EY1C8) established from peripheral blood lymphocytes obtained from a patient with aCL. RESULTS: Without APC, the formed thrombin activity decreased by the addition of 3.4 microM beta2GPI. When 12.8 nM APC was added, beta2GPI partially reversed the APC-induced inhibition of thrombin generation in a concentration-dependent manner. With 3.4 microM beta2GPI, the thrombin generation in monoclonal aCL (2.5 microg/ml) decreased to 77.1-80.2% by the addition of 12.8 nM APC, but the values were above that in the control IgM (72.7%). Without beta2GPI, the APC activity was unaffected by the addition of monoclonal aCL. CONCLUSION: Beta2-glycoprotein I exhibits procoagulant activity by inhibiting APC activity and anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL and only in the presence of beta2GPI.

Adenomatous Polyposis Coli Protein↗
PubMed1979

Microdensitometry.

Microdensitometry, or microspectrophotometry, is the measurement of the concentration or mass of a chromophore in microscopically defined regions, and is governed by well-established laws of physics. Initially it proved of value in Feulgen cytophotometry of the relative amounts of DNA in individual nuclei of isolated cells. It has now achieved wide applicability to the measurement of cellular biochemical activity by means of stoichiometric chromogenic reactions. The validity of some of these measurements has been confirmed by comparative biochemical and microdensitometric assays. Thus microdensitometry, even of heterogeneously distributed chromophores, can be precise, provided that the technique is operated with due regard to its limitations within the laws of physics. The potential errors include: variation in thickness of tissue sections (path-length); scatter; glare; diffraction; occlusion of light by optically dense particles; and the inhomogeneity error. However, under correct conditions for the cytochemical reactions and for operating the microdensitometer, these potential errors become small or negligible. Thus the highly sensitive cytochemical bioassay of thyrotropin exemplifies the precision that can be achieved by controlled use of microdensitometry.

Animals↗
PubMed1982

An assessment of the fibrinolytic system in hemophilia A.

The fibrinolytic system was assessed in 28 hemophiliacs using the chromogenic substrate H-D-Val-Leu-Lys-pNA. To determine whether a state of hyperfibrinolysis might be associated with Factor VIII replacement therapy, 14 patients with severe disease who were intensively treated with Factor VIII concentrates were compared with 14 patients with mild disease who were receiving infrequent transfusions with cryoprecipitate or fresh frozen plasma. Seventeen normal males served as controls. With the exception of an elevated level of plasminogen activator and a decreased level of immediate antiplasmin in the mild group only, no evidence of enhanced fibrinolysis was found. Other components of the fibrinolytic system were either normal (plasmin) or increased (progressive antiplasmin containing both alpha 2PI and alpha 2M, and plasminogen). The elevated plasminogen levels were found only in the severe intensively transfused group. The elevated progressive antiplasmin levels were found in both groups of patients and did not appear to be related to transfusions. These findings do not support the concept of enhanced fibrinolysis associated with intensive Factor VIII replacement therapy.

Adolescent↗
PubMed1989

Newborn's fibrinolytic mechanism: components and plasmin generation.

Plasminogen activity and antigen, tissue-type plasminogen activator (tPA) activity and antigen, plasminogen activator inhibitor (PAI) activity, and plasmin generation rates were determined in 32 normal newborn plasmas and 25 normal adult plasmas. The newborns showed reduced levels of plasminogen activity and antigen and tPA antigen, and activity, normal levels of PAI activity, and slower plasmin generation rates. The slower generation was shown to be due to the hypoplasminogenemia. The in vitro plasmin generation studies also showed that the newborn needed 11 times the usual concentration of urokinase and 5 times the usual concentration of tPA to achieve the minimal activation rate of the adult.

Caseins↗
PubMed2003

Thermodynamic determination of beta-hexosaminidase isoenzymes in mononuclear and polymorphonuclear leukocyte populations.

Isoenzymes of beta-hexosaminidase (Hex) were determined in mononuclear (MN) and polymorphonuclear (PMN) leukocytes, with a thermodynamic method using the chromogenic substrate sodio-3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide. Imprecision was very satisfactory, and the results are very much in agreement with those obtained using the fluorogenic substrates 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide 6-sulfate. In 163 healthy individuals we found, for the proportion as a percentage of the Hex A isoenzyme, significantly higher values (P < 0.001) in PMN than in MN cells (71.56 +/- 0.30% vs. 54.28 +/- 0.24%), meaning that it would not appear advisable to use total leukocyte lysates for evaluating this variable. The method is fast, precise, and highly suitable for the biochemical diagnosis and heterozygote screening of GM2 gangliosidoses, and would be applicable in cases of thermolabile Hex B and for detecting the B1 variant.

Adolescent↗
PubMed1984

Degradation of a chromogenic substrate by alpha 2-macroglobulin from plasma of patients with rheumatoid arthritis.

We have shown previously that serum from patients with rheumatoid arthritis (RA) contains a polyclonal B cell activator that is associated with alpha 2-macroglobulin (alpha 2M). Some biologic effects of this activator appear to be due to a trypsin-like protease attached to alpha 2M. Therefore, in the present study, we used an anti-alpha 2M antibody solid-phase assay, with Chromozym-Try as a substrate, to determine the level of alpha 2M-protease complexes in plasma alpha 2M. We found higher levels of these complexes in RA patients than in 2 control groups. Since alpha 2M-protease complexes have been shown to induce RA-like inflammation in experimental animals and to be produced by lymphoid cells, we speculate that they may be involved in the pathogenesis of RA. However, the role of the other cells or enzyme systems in the formation of these complexes has not yet been ruled out. Results of these investigations could lead to another link between activation of the immune system and joint inflammation.

Adult↗
PubMed2007

Demethyloleuropein and beta-glucosidase activity in olive fruits.

Demethyloleuropein plays a major role in the defense mechanism of olive fruits. To understand how this molecule is metabolized during different stages of maturation of olive fruits, a biomolecular approach to identify the demethyloleuropein chemistry was employed. The beta-glucosidase activity in crude extracts was assayed spectrophotometrically using the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Demethyloleuropein was extracted and identified by HPLC-MS from both infected and uninfected olive fruits at different physiological stages. The release of more functionally relevant dialdehydes in uninfected fruits was investigated using ESIMS/ MS. In fruits harvested in October, the activity of beta-glucosidase was significantly enhanced in uninfected fruits when compared to the infected fruits. Quantitative differences in the demethyloleuropein content from uninfected fruits showed the highest values (5.09 mg/g) in October, whereas lower levels (4.44 mg/g) were found in infected fruits. The results demonstrated that demethyloleuropein derivatives could be influenced by beta-glucosidase activity to improve the quality of the olive products with the best dialdehyde nutraceutical content.

Animals↗
PubMed2003

Determination of laccase activity in mixed solvents: comparison between two chromogens in a spectrophotometric assay.

Spectrophotometric determination of laccase activity with ABTS acting as chromogen yields exceedingly low values whenever conducted in a water-organic mixed solvent. Nevertheless, there is firm evidence that laccase is able to oxidize substrates such as phenols and amines quantitatively in these mixed solvents. We show that the apparently small rate of ABTS oxidation by laccase in a mixed solvent, such as buffered water-dioxane 1:1, is not amenable to the denaturation of laccase but rather to the decreased stability of ABTS(.+). We propose HAA as a more reliable chromogen for the determination of laccase activity in mixed solvents.

3-Hydroxyanthranilic Acid↗
PubMed2006

Enhanced molecular recognition signal in allosteric biosensing by proper substrate selection.

Among protein biosensors, those based on enzymatic responses to specific analytes offer convenient instruments for fast and ultra-fast molecular diagnosis, through the comparative analysis of the product formed in presence and in absence of the effector. We have explored here the performance of five beta-galactosidase substrates during the activation of a beta-galactosidase sensor by antibodies against the human immunodeficiency virus (HIV). Interestingly, the employed substrate determines the dynamic range of the allosteric signal and significantly influences the sensitivity of the senso-enzymatic reaction. While ortho-nitrophenyl beta-D-galactopyranoside allows the detection of a model anti-gp41 monoclonal antibody below 0.024 ng/microL, phenol red beta-D-galactopyranoside offers the most dynamic response with signal/background ratios higher than 12-fold and a detection limit around 0.071 ng/microL. The hydrolysis of both chromogenic substrates generates linear sensing responses to immune human sera and parallel time-course topologies of the allosteric reaction. Therefore, the obtained results stress the potential of chromogenic substrates versus those rendering quimioluminescent, amperometric, or fluorescent signals, for the further automatization, miniaturization, or adaptation of beta-galactosidase-based biosensing to high-throughput applications.

Allosteric Regulation↗