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PubMed2004

Chromogenic charge transfer cleft-type tetrahydrobenzoxanthene enantioselective receptors for dinitrobenzoylamino acids.

A combination of a benzoxanthene cleft-type receptor with an electron-rich aromatic ring capable of establishing charge-tranfer interactions provides enantioselective receptors for dinitrobenzoylamino acids. Racemic mixtures of the receptor can be resolved with TLCs impregnated with the guest. The structure of the complexes has been established in an X-ray study. Enantiomeric amino acids provide complexes with different colors.

Chromogenic Compounds↗
PubMed2006

Mechanistic aspects of proton chain transfer: a computational study for the green fluorescent protein chromophore.

We explore several models for the ground-state proton chain transfer pathway between the green fluorescent protein chromophore and its surrounding protein matrix, with a view to elucidating mechanistic aspects of this process. We have computed quantum chemically the minimum energy pathways (MEPs) in the ground electronic state for one-, two-, and three-proton models of the chain transfer. There are no stable intermediates for our models, indicating that the proton chain transfer is likely to be a single, concerted kinetic step. However, despite the concerted nature of the overall energy profile, a more detailed analysis of the MEPs reveals clear evidence of sequential movement of protons in the chain. The ground-state proton chain transfer does not appear to be driven by the movement of the phenolic proton off the chromophore onto the neutral water bridge. Rather, this proton is the last of the three protons in the chain to move. We find that the first proton movement is from the bridging Ser205 moiety to the accepting Glu222 group. This is followed by the second proton moving from the bridging water to the Ser205--for our model this is where the barrier occurs. The phenolic proton on the chromophore is hence the last in the chain to move, transferring to a bridging "water" that already has substantial negative charge.

Chromogenic Compounds↗
PubMed2006

Gauging the effect of impurities on lipid bilayer phase transition temperature.

We report on the gel-to-fluid phase transition behavior of unilamellar vesicles formed with 1,2-dimyristoyl-sn-phosphatidylcholine (14:0 DMPC). We have interrogated the gel-to-fluid transition temperature of these bilayer structures using the chromophore perylene incorporated in their nonpolar region. We observe a discontinuous change in the reorientation time of perylene sequestered within the bilayer at the known melting transition temperature of 14:0 DMPC, 24 degrees C. The perylene reorientation data reveal a local viscosity of 14.5 +/- 2.5 cP in the gel phase, and 8.5 +/- 1.5 cP in the fluid phase. We have also incorporated small amounts of 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (14:1 DMPC) into these unilamellar vesicles and find that the melting transition temperature for these bilayers varies in a regular manner with the amount of 14:1 DMPC present. These data demonstrate that very little "contaminant" is required to cause a substantial change in the gel-to-fluid transition temperature, even though these contaminants do not alter the viscosity of the bilayer sensed by perylene, either above or below the melting transition.

Chromogenic Compounds↗
PubMed2006

Hidden Markov model analysis of multichromophore photobleaching.

The interpretation of single-molecule measurements is greatly complicated by the presence of multiple fluorescent labels. However, many molecular systems of interest consist of multiple interacting components. We investigate this issue using multiply labeled dextran polymers that we intentionally photobleach to the background on a single-molecule basis. Hidden Markov models allow for unsupervised analysis of the data to determine the number of fluorescent subunits involved in the fluorescence intermittency of the 6-carboxy-tetramethylrhodamine labels by counting the discrete steps in fluorescence intensity. The Bayes information criterion allows us to distinguish between hidden Markov models that differ by the number of states, that is, the number of fluorescent molecules. We determine information-theoretical limits and show via Monte Carlo simulations that the hidden Markov model analysis approaches these theoretical limits. This technique has resolving power of one fluorescing unit up to as many as 30 fluorescent dyes with the appropriate choice of dye and adequate detection capability. We discuss the general utility of this method for determining aggregation-state distributions as could appear in many biologically important systems and its adaptability to general photometric experiments.

Chromogenic Compounds↗
PubMed2005

Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots.

This report describes two related methods for decorating cowpea mosaic virus (CPMV) with luminescent semiconductor nanocrystals (quantum dots, QDs). Variants of CPMV are immobilized on a substrate functionalized with NeutrAvidin using modifications of biotin-avidin binding chemistry in combination with metal affinity coordination. For example, using CPMV mutants expressing available 6-histidine sequences inserted at loops on the viral coat protein, we show that these virus particles can be specifically immobilized on NeutrAvidin functionalized substrates in a controlled fashion via metal-affinity coordination. To accomplish this, a hetero-bifunctional biotin-NTA moiety, activated with nickel, is used as the linker for surface immobilization of CPMV (bridging the CPMVs' histidines to the NeutrAvidin). Two linking chemistries are then employed to achieve CPMV decoration with hydrophilic CdSe-ZnS core-shell QDs; they target the histidine or lysine residues on the exterior virus surface and utilize biotin-avidin interactions. In the first scheme, QDs are immobilized on the surface-tethered CPMV via electrostatic attachment to avidin previously bound to the virus particle. In the second strategy, the lysine residues common to each viral surface asymmetric unit are chemically functionalized with biotin groups and the biotinylated CPMV is discretely immobilized onto the substrate via NeutrAvidin-biotin interactions. The biotin units on the upper exposed surface of the immobilized CPMV then serve as capture sites for QDs conjugated with a mixture of avidin and a second protein, maltose binding protein, which is also used for QD-protein conjugate purification. Characterization of the assembled CPMV and QD structures is presented, and the potential uses for protein-coated QDs functionalized onto this symmetrical virion nanoscaffold are discussed.

Chromogenic Compounds↗
PubMed1998

Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation.

Under acidic and mild-temperature conditions, 1-methyl-2-phenylindole was found to react with malondialdehyde (MDA) and 4-hydroxyalkenals to yield a stable chromophore with intense maximal absorbance at 586 nm. The use of methanesulfonic acid results in optimal yields of chromophore produced from MDA as well as from 4-hydroxynonenal. By contrast, the use of hydrochloric acid results in an optimal yield of chromophore produced from MDA and a negligible reaction of 4-hydroxynonenal. Taking advantage of such chromogenic reactions, we developed a new colorimetric assay of lipid peroxidation. Using a methanesulfonic acid-based medium, MDA and 4-hydroxyalkenals can be measured at the 586 nm wavelength. However, the presence of endogenous inhibitors of the reaction with 4-hydroxyalkenals is common, and this means that the latter may be underestimated in some biological samples. The assay performed in a hydrochloric acid-based medium enables the specific measurement of MDA in the presence of 4-hydroxyalkenals. Upon hydrolysis of Schiff bases in hydrochloric acid (pH 1.5), either assay can be used to specifically measure the amount of total MDA in biological samples because 4-hydroxyalkenals undergo an irreversible cyclization reaction under the hydrochloric acid-based conditions of hydrolysis. The two assays were applied to the determination of the amount of MDA alone and of MDA and 4-hydroxyalkenals in an in vitro model of lipid peroxidation. This methodology was also used to clarify complex patterns of tissue-specific MDA production in vivo, following hydrolysis of Schiff bases, in rodents treated with doxorubicin.

Aldehydes↗
PubMed1998

Reactions of N-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Mechanistic aspects of the colorimetric assay of lipid peroxidation.

Under specific acidic conditions, both malondialdehyde (1, MDA) and 4-hydroxynonenal (2, 4-HNE) react with N-methyl-2-phenylindole (3) to give the same chromophoric cyanine 4 with maximal absorbance at 586 nm. Under such conditions, the reaction of 3 with 4-HNE (2) as well as with alkanals yields a second chromophoric cyanine 10 with maximal absorbance at 505 nm. The influence of different acids, iron(III), and oxygen on the reaction of 3 with such aldehydes was studied in detail. Under anaerobic conditions, the acid-induced reaction of 4-HNE with 3 afforded three rapidly interconverting intermediates, 5-7. Their subsequent fragmentation to 4 and hexanal in the presence of iron(III) and oxygen is consistent with the tandem beta-fragmentation of an indolyl radical cation. 1-Indolylalkenes were identified as essential intermediates in the acid-induced reaction of 3 with alkanals. A very mild iron(III)-catalyzed fragmentation of these intermediates afforded the corresponding 3-formylindole 11 as the direct precursor of the 505 nm chromophore 10. Such reactions were markedly influenced by the nature of the acid. Contrary to the rapid chromogenic reaction of 4-HNE which was observed in the presence of methanesulfonic acid, the HCl-induced reaction of 4-HNE with 3 did not afford the 586 nm chromophore. Furthermore, hexanal did not yield the 505 nm chromophore 10 upon reaction with 3 in the presence of HCl, again in contrast with the rapid chromogenic reaction which was observed in the presence of methanesulfonic acid. Comparison of the reaction mixtures under the two assay conditions confirmed that the same intermediates were formed. We conclude that the nature of the acid plays a crucial role in the oxidative fragmentation of intermediates into chromophores, allowing the selective assay of MDA in the presence of 4-HNE, using HCl acidic conditions.

Aldehydes↗
PubMed1997

Bovine erythrocyte haemolysates enhance plasminogen activation by tissue-type plasminogen activator.

An active fraction that accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) was purified from a haemolysate of bovine erythrocytes. When the haemolysate was mixed with t-PA, it produced a 2- to 3-fold increase in plasminogen activation as measured by an insoluble fibrinolytic assay system and a soluble amidolytic assay system with the chromogenic substrate S-2251. Zymographic analysis showed that, while the haemolysate increased t-PA activity, it did not alter the electrophoretic characteristics of the t-PA nor did it induce any fibrinolysis in the absence of t-PA or plasminogen. The haemolysate was devoid of plasmin and plasminogen activator activity but was most effective in accelerating plasminogen activation by t-PA in the presence of substrate. Based on the purification characteristics of the active fraction in the haemolysate, it appears to have a molecular weight of less than 10 kDa.

Animals↗
PubMed1997

Appearance of colonies of Prototheca on CHROMagar Candida medium.

The microorganisms capable of producing opportunist infections include the yeast-like organisms of the genus Candida, and the unicellular algae of the genus Prototheca, which share common features and can, therefore, lead to confusion. Their colonies are almost identical and they grow in the same culture media used routinely in mycology. CHROMagar Candida is a new chromogenic differential isolation medium that facilitates the presumptive differentiation of some of the most clinically important yeast-like organisms. To our knowledge, the use of CHROMagar Candida with Prototheca spp. has not been reported in the literature. This report describes the growth of 151 strains of Prototheca on CHROMagar Candida compared to the growth of a total of 326 well-characterized yeast organisms of the genera Candida, Cryptococcus, Trichosporon, Geotrichum, and Saccharomyces. It is clinically relevant to note that algae of the genus Prototheca (P. wickerhamii, P. zopfii, and P. stagnora) and of the genus Candida parapsilosis produced similar cream-colored colonies on CHROMagar Candida medium. Based on their growth on CHROMagar, a new species of Candida is described, C. zeylanoides, which has blue-green colonies. The colonies of two species of Trichosporon are also differentiated: the blue-green colonies of T. beigelii and the pink colonies of T. capitatum.

Candida↗
PubMed2001

Peroxidase-catalyzed co-oxidation of 3,3',5,5'-tetramethylbenzidine with 2-amino-4-nitrophenol, 4,4'-dihydroxydiphenylsulfone, and their polydisulfides in aqueous and micellar media.

The effects of different concentrations of 2-amino-4-nitrophenol (ANP) and of its polydisulfide (poly(ADSNP)) on peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) were studied at 20 degrees C in reversed micelles of AOT (0.2 M) in heptane and in mixed reversed micelles of AOT (0.1 M)-Triton X-100 (0.1 M) in isooctane supplemented with 15% hexanol. The oxidation of TMB was activated nearly twofold in the presence of ANP and nearly fourfold in the presence of poly(ADSNP) in reversed micelles of AOT, whereas in the mixed micelles oxidation of the TMB-ANP pair was associated with inhibition of TMB conversion and poly(ADSNP) activated oxidation of TMB. The co-oxidation of TMB with 4,4'-dihydroxydiphenylsulfone (DDS) and with its polydisulfide (poly(DSDDS)) at different concentrations of phenol components was accompanied by activation of TMB conversion in 0.01 M phosphate buffer (pH 6.4) supplemented with 5% DMF and in reversed micelles of AOT in heptane. The effect of pH of the aqueous solution on the initial oxidation rate of the TMB-DDS and TMB-poly(DSDDS) pairs and also the effect of hydration degree of reversed micelles of AOT on conversion of the same pairs by peroxidase were studied. A scheme of peroxidase-dependent co-oxidation of "aromatic amine-phenol" pairs is proposed and discussed. A significant part of this scheme is a nonenzymatic exchange of phenoxyl radicals with amines and of aminyl radicals with phenols.

Benzidines↗
PubMed2001

Butyrylcholinesterase-Mediated enhancement of the enzymatic activity of trypsin.

1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are enzymes that catalyze the hydrolysis of esters of choline. 2. Both AChE and BuChE have been shown to copurify with peptidases. 3. BuChE has also been shown to copurify with other proteins such as transferrin, with which it forms a stable complex. In addition, BuChE is found in association with beta-amyloid protein in Alzheimer brain tissues. 4. Since BuChE copurifies with peptidases, we hypothesized that BuChE interacts with these enzymes and that this association had an influence on their catalytic activities. One of the peptidases that copurifies with cholinesterases has specificity similar to trypsin, hence, this enzyme was used as a model to test this hypothesis. 5. Purified BuChE causes a concentration-dependent enhancement of the catalytic activity of trypsin while trypsin does not influence the catalytic activity of BuChE. 6. We suggest that, in addition to its esterase activity, BuChE may assume a regulatory role by interacting with other proteins.

Acetylcholinesterase↗
PubMed2002

A new assay to monitor the degranulation process in phagocytizing human neutrophils.

oxidation, we set up a method for measuring MPO intraphagosomal release in human neutrophils. The method is based on the passive engulfment of DAB together with the phagocytosable particle. Inside the vacuole, this substrate is oxidized by MPO released from the azurophilic granules. The colorimetrical evaluation of the amount of DAB oxidized allows for cheap, rapid quantification of MPO intraphagosomal secretion in whole cells. Using this method, we show that the degranulation process, involving azurophilic granules, can be monitored carefully during phagocytosis. It takes place after the ingestion of zymosan particles opsonized with normal human serum, as well as during IgG-mediated phagocytosis and under conditions where beta2 integrins are blocked. However our findings also show that the extent of intraphagosomal secretion depends on either the extent of opsonization or the type of receptor engaged during the phagocytic event.

3,3'-Diaminobenzidine↗
PubMed2002

Suppression of matrix metalloproteinase activity by SI-27: detection by a new activity assay with S-2444, a specific chromogenic peptide.

We have previously reported on the anti-invasive and angiosuppressive effects of SI-27, an anti-matrix metalloproteinase (MMP) agent. The molecular mechanism of its anti-MMP action, however, has not yet been determined. The purpose of this study was to investigate the effects of SI-27 on MMP- 1, -2, -3, -9, and TIMP-1, -2 secreted by human glioma cell lines (U87MG, U251MG, U373MG, and Y98G). When cells were exposed to non-cytotoxic concentrations of SI-27 (preliminarily determined by the MTT assay), expressions of mRNAs for the enzymes was not inhibited. For an MMP activity assay, we employed the fact that active MMPs could cleave modified pro-urokinase to form active urokinase, which then acted on S-2444 peptide to create a chromogenic product. Secretion of all pro-MMPs from glioma cells was not significantly reduced by SI-27. However, activation of pro-MMPs was significantly inhibited in a dose-dependent manner ((IC50 values for MMP-2; U87MG, 3.5 microg/ml; U25 IMG, 4.2 microg/ml; U373MG, 4.8 microg/ml; Y98G, 4.0 degreesg/ml); (IC50 values for MMP-9; 251MG, 7.2 microg/ml, U373MG, 2.8 microg/ml). In addition, active MMPs were not inhibited by SI-27. These findings were supported by zymographic analysis and by collagenolysis assay data. TIMP-1 and -2 were also not inactivated by SI-27. These findings suggest that SI-27 targets the activation process of pro-MMP. S-2444, a specific chromogenic peptide, was useful for quantitative analysis of the activity of MMP subtypes in this study.

Chromogenic Compounds↗
PubMed2002

Comparative study of fluorogenic and chromogenic media for specific detection of environmental isolates of thermotolerant Escherichia coli.

In a field study 78 water samples were analysed employing Fluorocult Brilla Broth (BB) and its performance was compared with standard MPN procedure. Out of 78 water samples analysed 56 (71.7%) samples yielded positive reactions in BB whereas, 50 (64.1%) samples were positive by standard fecal coliform test. A comparative study of fluorogenic and chromogenic media containing substrate beta-D glucuronide for specific detection of environmental isolates of 313 thermotolerant E. coli has been undertaken. Five fluorogenic media were used: Fluorocult MacConkey agar (MCA), Fluorocult ECD agar (ECD), Fluorocult VRB agar (VRB), Fluorocult E. coli 0157:H7 agar (ECH7) and Fluorocult Brilla Broth (BB) and Chromogenic Chromocult agar (CCA). BB and CCA were found to be highly specific and sensitive media to detect E. coli as all E. coli yielded positive reaction on them. On ECH7 and ECD agar 67.5 and 64.9 of E. coli isolates gave positive reaction, respectively. Low sensitivity was observed in case of MCA and VRB agar in detecting E. coli. The performance of BB appears to be better when compared with standard MPN procedure employing MacConkey broth/Brilliant green bile broth in detecting E. coli in drinking water.

Adaptation, Physiological↗
PubMed1999

The proteolytic activity and cleavage specificity of fibronectin-gelatinase and fibronectin-lamininase.

Human plasma fibronectin contains two latent aspartic proteinases, FN-gelatinase and FN-lamininase. Both enzymes can be generated and activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment. We investigated the proteolytic activity and cleavage specificity of both enzymes in a range of pH from 3.5 to 9.0 using the B chain of oxidized bovine insulin and chromogenic peptides as substrates. The inhibition of the enzymes by several natural inhibitors from human plasma was also tested. The specificities of FN-gelatinase and FN-lamininase are similar to other major acidic proteinases, including pepsin, renin, cathepsin D, and HIV-proteinases. Both enzymes mainly hydrolyze three peptide bonds in the oxidized insulin B chain, namely Glu-Ala (residues 13-14), Tyr-Leu (residues 16-17), and Phe-Phe (residues 24-25). For the peptide substrates H-Pro-Thr-Glu-Phe-p-nitro-Phe-Arg-Leu-OH and H-Phe-Gly-His-p-nitro-Phe-Phe-Val-Leu-OMe that were cleaved the respective values of k(cat)/K(M) were 105.1 and 11.8 mM(-1) sec(-1) for cleavage by FN-gelatinase, and 123.2 and 15.5 mM(-1) sec(-1) for cleavage by FN-lamininase. The maximal activities of both enzymes were observed in a range between pH 5.6 and 6.3 and they became inactivated at a pH value above 8.4. Both FN-gelatinase and FN-lamininase were efficiently inhibited by alpha2-macroglobulin.

Calcium↗
PubMed2003

Activities of anionic and cationic trypsins in the temperature range from 5 to 37 degrees C. Mutant anionic trypsins as a model of cold-adapted (psychrophilic) enzymes.

Temperature dependences of kinetic constants (k(cat) and K(m)) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of cold-adapted fishes. The anionic and cationic trypsins were found to differ in organization of the S(1)-substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S(1)) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K(m) value) to the anionic and cationic trypsins and on the catalytic activity k(cat) should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.

Animals↗
PubMed2001

Enzymatic synthesis of chromogenic substrates for Glu,Asp-specific proteinases.

Glu,Asp-specific endopeptidases represent a new subfamily of chymotrypsin-like proteolytic enzymes. These enzymes prefer Glu or Asp residues in the P1 position of the substrates. p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal glutamic or aspartic acid residues have been obtained. Acyl peptide p-nitroanilides were synthesized via acylation of glutamic or aspartic acid p-nitroanilides using methyl esters of the respective N-acylated peptides, generally with good yields. The reactions were performed in organic solvents using subtilisin 72 sorbed on silica as a catalyst. The kinetic parameters for the hydrolysis of these p-nitroanilides with proteinases from Bacillus intermedius and Bacillus licheniformis were determined.

Anilides↗