[ISOLATION IN URINES OF PREGNANT WOMEN OF 5 COMPOUNDS CHROMOGENIC TO TETRAZOLIUM BLUE, ABSORBANT AT 254 MILLIMICRONS, AND FLUORESCENT IN SODIUM MEDIUM].
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Adrenal Cortex Hormones↗
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Results for “Chromogenic Compounds”
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Purification of commercial coomassie brilliant blue R-250 and characterization of the chromogenic fractions.
Coomassie brilliant blue R-250 (CBB) is a popular and widely used dye for detection of proteins by gel electrophoresis. However, commercially available CBBs are complex mixtures of numerous chromogenic compounds that vary from lot to lot, thereby giving an undesirable level of variation in reproducibility, precision, and specificity in staining gels. We have developed a silica gel column chromatographic method for purification of commercial CBBs in high yield and have standardized each lot to perform equivalently in staining proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitative scanning densitometry. This is a major improvement in protein purity determinations by quantitative scanning densitometry. A thinlayer chromatographic method for quality control testing of the purified CBB lots was also developed. Plasma desorption mass spectrometry was used to identify components of silica gel column fractions. Scanning densitometry was the technology used to establish performance equivalency between different CBB preparations. The less polar chromogenic compounds are nonblue and/or fluorescent in color, contain mono- or unsulfonated structures, and lack significant protein binding capacity. The more polar chromogenic compounds are green and blue-green in color, contain tri- and tetrasulfonated moieties, compared to the disulfonated structure of CBB, and bind to protein at least 40 times more effectively than pure CBB. The concentrations of these highly polar chromogens differ from lot to lot and act as "inhibitors" in protein staining, thereby causing variability in protein staining.
Chromatography, Thin Layer↗
Novel compound for identifying Escherichia coli.
A new chromogenic compound, 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide, was found to be useful for the rapid, specific, differential identification of Escherichia coli in the sanitary analysis of shellfish and wastewater. Of 1,025 presumptively positive colonies (blue) and 583 presumptively negative colonies (nonblue), only 1% false-negative and 5% false-positive results were found.
Animals↗
[Dynamic changes of the plasma prekallikrein activity in patients with acute myocardial infarction].
The activity of the plasma prekallikrein was assayed by using dextran sulfate as activator and chromogenic compound substance Na-benzoyl-DL-arginine-4-nitroanilid-hydrochloride (BAPNA) as substrate. The dynamic changes of the plasma prekallikrein activity were observed in 22 patients with acute myocardial infarction (AMI) at different period. The correlation between plasma prekallikrein activity and serum CPK activity were analysed in part of the patients. The results indicate that the plasma prekallikrein activity decreased significantly in AMI, especially on the third day after onset. The decrease of plasma prekallikrein and increase of serum CPK were correlated negatively. The results indicate that the chromogenic compound substance BAPNA can be used to determine the activity of plasma prekallikrein. The kinin system was activated by certain factors in AMI, causing the decrease of plasma prekallikrein and increase of bradykinin.
Adult↗
Chromogenic derivatives of AHTN (6-acetyl-1,1,2,4,4,7-hexamethyltetralin) react with amino acids and protein in vitro. Spectral characteristics of the colour products.
Three chromogenic products of AHTN have been detected after photo-oxidation and shown to react with glycine or albumin, giving rise first to a blue color, followed by pink and, finally, a dark green colour. The absorption spectrum associated with the late green colour showed an increased absorbance in the long wavelength region of the spectrum, similar to the green colour extracted from the liver of AHTN-treated animals. The colour produced by the chromogenic derivatives of AHTN and by o-diacetylbenzene (a model chromogenic compound) were tightly bound to the protein pellet and resistant to acidic pH, unlike the colour from the ninhydrin reaction. The dark green colour produced in the liver by feeding AHTN to rats was also tightly bound to proteins and stable to acidic pH. These results suggest that both the photo-oxidized chromogenic derivatives of AHTN and o-diacetylbenzene, produce coloured derivatives with amino acids and proteins by a mechanism unrelated to ninhydrin. They also suggest that a chromogenic derivative of AHTN, produced in vivo during prolonged treatment with high doses of AHTN, may be responsible for the green colour of the livers and other tissues from treated animals. The data suggest that the AHTN-derived chromogenic material is metabolite-related rather than representative of a toxic process, although further work is necessary to confirm this hypothesis.
Amino Acids↗
Genetic and biochemical characterization of the pathway in Pantoea citrea leading to pink disease of pineapple.
Pink disease of pineapple, caused by Pantoea citrea, is characterized by a dark coloration on fruit slices after autoclaving. This coloration is initiated by the oxidation of glucose to gluconate, which is followed by further oxidation of gluconate to as yet unknown chromogenic compounds. To elucidate the biochemical pathway leading to pink disease, we generated six coloration-defective mutants of P. citrea that were still able to oxidize glucose into gluconate. Three mutants were found to be affected in genes involved in the biogenesis of c-type cytochromes, which are known for their role as specific electron acceptors linked to dehydrogenase activities. Three additional mutants were affected in different genes within an operon that probably encodes a 2-ketogluconate dehydrogenase protein. These six mutants were found to be unable to oxidize gluconate or 2-ketogluconate, resulting in an inability to produce the compound 2,5-diketogluconate (2,5-DKG). Thus, the production of 2,5-DKG by P. citrea appears to be responsible for the dark color characteristic of the pink disease of pineapple.
Chromogenic Compounds↗
Tay-Sachs disease: one-step assay of beta-N-acetylhexosaminidase in serum with a sulphated chromogenic substrate.
A sulphated chromogenic compound, p-nitrophenyl-6-sulpho-2-acetamido-2-deoxy-beta-D-glucopyranoside, which can be hydrolysed enzymatically to p-nitrophenol and the sulphated amino sugar, was used as a substrate for the determination of activity of beta-N-acetylhexosaminidase isoenzymes in human serum. The sera of six Tay-Sachs patients lacking isoenzyme A and heat-inactivated control serum exhibited 6% of the mean normal enzyme activity of 1.32 U/l (1-s range = 1.07-1.57 U/l). In 10 obligate carriers of the Tay-Sachs gene the enzyme activity was 52% (1-s range = 45-60%) of the mean normal value. Therefore, by using the sulphated chromogenic substrate Tay-Sachs disease can be diagnosed enzymatically in a simple one-step procedure, but the 2-s activity ranges of heterozygotes and normals overlap. The assay is not absolutely specific for isoenzyme A of beta-N-acetylhexosaminidase, because the substrate can be hydrolysed to a certain extent by beta-N-acetylhexosaminidase I.
Acetylglucosamine↗
[A combined chromogenic-fluorogenic medium for the simultaneous detection of coliform groups and E. coli in water].
A comparison was made with different chromogenic and fluorogenic substrates, 4-methylumbelliferyl-beta-D-glucuronide (MUG), 4-nitrophenyl-beta-D-glucuronide (PNPG), 4-methylumbelliferyl-beta-D-galactopyranoside (MUGA), 2-nitrophenyl-beta-D-galactopyranoside (ONPG), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL), for the rapid and simultaneous enumeration of total coliforms and E. coli in water samples, based on 2 commercially available culture-media. The combination of the chromogenic compound X-GAL (for detecting coliforms) and of the fluorogenic compound MUG (for detecting E. coli) incorporated either into ECD agar or into lauryl sulfate broth proved to be most useful. The optimum concentration of the X-GAL/MUG supplement was (50 micrograms/ml/70 micrograms/ml) for the solid medium (EMX agar) and (60 micrograms/ml/70 micrograms/ml) for the fluid medium (LMX broth). As a result of the examination of 244 Enterobacteriaceae strains isolated from water samples and clinical material, it was shown that the use of EMX agar (LMX broth) had several advantages over conventional methods. A routine method for the analysis of water samples was proposed involving the EMX agar and the LMX broth.
Chromogenic Compounds↗
Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue.
We developed a new, highly sensitive enzymatic method for quantifying creatine in erythrocytes, which comprises creatine amidinohydrolase, sarcosine oxidase, and peroxidase. In the present method, an N-methylcarbamoyl derivative of methylene blue, 10-N-methylcarbamoyl-3,7-bis(dimethylamino)phenothiazine (MCDP), was used as a sensitive chromogenic compound. Potassium ferrocyanide was used to prevent nonspecific oxidation of MCDP. The enzymatic method exhibited good analytical performance: precision, within-run CVs <1.0% and between-day CVs <2.0%; average analytical recovery, 99.3% +/- 1.8%; detection limit, 1.0 micromol/L in hemolysate; and linearity, at least up to 500 micromol/L as creatine concentration in hemolysate. Excellent agreement was observed between the present method (y) and HPLC (x), y = 1.029x - 0.002 micromol/g hemoglobin, r = 0.9998, S(y/x) = 0.053 micromol/g hemoglobin (n = 110). No significant interference was produced by various compounds, including guanidino compounds, amino acids, and reducing materials. The reference intervals (mean +/- 2 SD) for erythrocyte creatine obtained from 60 males and 60 females were (in micromol/g hemoglobin) 1.18 +/- 0.52 (0.66-1.70) for males and 1.35 +/- 0.49 (0.86-1.84) for females. Using this method, we documented changes in erythrocyte creatine in patients with various hemolytic conditions, including hemolytic anemia, liver cirrhosis, renal insufficiency, and chronic renal failure treated with hemodialysis with or without the administration of erythropoietin. We conclude that the use of MCDP allows sensitive measurement of erythrocyte creatine and that MCDP with potassium ferrocyanide can improve the sensitivity of assays that use peroxidase for detection of H2O2.
Adult↗
Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectivity and site-specific inhibitor determination.
The IQ Technology has been developed to serve as a homogeneous, universal detection platform for HTS of kinases and phosphatases. The technology is a direct, noncompetitive assay format that does not require antibodies or radioactive reagents to measure phosphorylation state. Fluorophore-labeled peptides are used as enzyme substrates, and kinase or phosphatase activity is quantitated by direct measurement of the phosphorylation state of the substrate. Phosphorylation is measured by the change in fluorescence intensity that occurs when a proprietary iron-containing compound binds specifically to phosphoryl groups on peptides. This change in observed fluorescence is proportional to the extent of phosphorylation of the fluorophore-labeled peptide. The technology provides a universal method that can be used with any peptide sequence and is insensitive to high concentrations of ATP. Inhibition at the ATP-binding site versus the phosphorylation site can be differentiated and compound selectivity identified using the same detection method as in the primary screen. The technology has been tested against a large number of detergents, organics, and other reagents found in reaction mixtures, and the detection method eliminates common issues associated with fluorescent and chromogenic compounds. The technology has been formatted for 96-, 384-, and 1,536-well microplate formats, and a representative Z' value of 0.7 was obtained. IC(50) values generated using this platform correlate with previously reported values, and screening of a small compound library was performed to evaluate the assay further.
Amino Acid Sequence↗
Taipan viper venom and chromogenic substrate (chromozym Th). Prothrombin assay. No sensitivity to coumarin-induced prothrombin.
Prothrombin (factor II) was assayed in congenital or acquired prothrombin deficiencies and abnormalities using Taipan viper (Oxyuranus scutellatus) venom as activating agent and adsorbed normal plasma or a chromogenic compound (Chromozym-Th) as substrates. Prothrombin was found to be low, as expected in every instance, regardless of the substrate used. In coumarin treated patients the levels observed were similar to the prothrombin time percentile values and definitely lower than the immunological counterparts. Therefore, the Taipan viper venom appears suited for prothrombin assay even during anticoagulant therapy. Since the cost of the chromogenic substrate is about 20 times that of adsorbed normal plasma and since no special information is obtained by it, the use of the chromogenic method is not justified for routine purposes.
Blood Coagulation Disorders↗
Azobenzene as conformational switch in model peptides.
The photoinduced isomerization of azobenzene between the extended (trans) and compact (cis) conformations is reversibly triggered by light of two differing wavelengths. The resulting changes in molecular geometry have been extensively utilized to photoswitch transformations in chemical species reversibly for applications in optoelectronic devises as well as to photocontrol conformational states in (bio)polymers. The high isomerization yield, remarkable photostability and ultrafast kinetics (few ps) of azobenzene are well suited for the design of small, defined model systems that allow detailed folding studies to be carried out both experimentally and theoretically on the same molecules. In our and other laboratories such systems were recently obtained with cyclic peptides of defined conformational preferences as well as with alpha-helical and beta-hairpin peptides. These should, by comparison of simulation and experiment, permit an assessment and improvement of the theoretical description on the one hand and a detailed interpretation of the ultrafast conformational dynamics on the other. The phototriggered changes in conformational states lead to concurrent changes in biophysical properties that can be exploited in the photocontrol of biochemical and biological events, as exemplarily discussed with redox-active cyclic bis-cysteinyl peptides and receptor ligands.
Azo Compounds↗
A new chromophoric assay for arginase activity.
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Aminocaproates↗
Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase.
This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitates outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performed in situ.
Acid Phosphatase↗
Characterization of the preparation process and the photochemical control of electrical properties of bilayer lipid membranes containing azobenzene chromophores.
We prepared photoresponsive bilayer lipid membranes (BLMs) containing azobenzene derivatives (4'-octylazobenzene-4-oxybutyric acid (AZ)) and observed the rapid and reversible changes in their electrical properties when irradiated with light. The BLMs consist of AZ (8 mol%) and glyceryl monooleate. The changes in capacitance and conductance upon irradiation by light were found to be 10 and 20%, respectively. The changes in the electrical properties of the membrane and in the structure of AZ under light irradiation were analyzed simultaneously by in-situ spectroscopic, electrical and microscopic measurements. These measurements showed that the electrical changes induced by exposure to light resulted from reversible changes in the membrane structure initiated by the photoisomerization reaction of AZ. This structural change in the membrane occurred within 1 s.
Azo Compounds↗
Differentiation of thrombin- and factor Xa-related amidolytic activity in plasma by means of a synthetic thrombin inhibitor.
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Aniline Compounds↗
Hydrolysis of muscle myofibrillar proteins by Lactobacillus curvatus and Lactobacillus sake.
Proteolytic enzyme activities of whole cells and cell free extracts (CFE) of Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were characterised using synthetic chromogenic compounds and myofibrillar proteins as substrates. The hydrolytic action was monitored by SDS-PAGE and reverse phase-HPLC analyses. The CFE of L. sake partially contributed, together with muscle enzymes, to the initial hydrolysis of myofibrillar proteins. Whole-cells of both L. curvatus and L. sake generated peptides considered important for cured-meat taste. The peptide mapping, resulting from the action on the substrates assayed, revealed a profile of extra and intracellular enzymes. Both strains expressed strong amino acid metabolism.
Aminopeptidases↗
Influence of the thickness of mineral trioxide aggregate on sealing ability of root-end fillings in vitro.
The purpose of this study was to compare the ability of different thicknesses of mineral trioxide aggregate (MTA) to prevent apical leakage through the use of a protein-dye complex with Coomassie Brilliant Blue G. Sixty-four teeth were divided into 4 groups, then filled with MTA to depths of 1, 2, 3, or 4 mm. Two teeth served as the positive controls. Another 2 teeth served as the negative controls. Root-filled teeth were mounted in an apparatus and then challenged with protein solution. The evaluation was conduced at 24-hour intervals for 60 days. The 1-mm-thick MTA was the least effective in preventing apical leakage (P <.05). No significance difference was found between 2- and 3-mm-thick MTA (P >.05). Four-millimeter-thick MTA was significantly more effective than the other thicknesses tested (P <.05). The results of this study suggest that the thickness of 4 mm is most adequate for the use of MTA as a root-end filling material.
Aluminum Compounds↗