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At least 37 records · Page 2Linked to original sources
PubMed1989

Protection of hexaprenyl-diphosphate synthase of Micrococcus luteus B-P 26 against inactivation by sulphydryl reagents and arginine-specific reagents.

Hexaprenyl-diphosphate synthase from Micrococcus luteus B-P 26 has been shown to comprise two essential components, designated as components A and B. Treatment of the synthase with sulphydryl reagents (N-ethylmaleimide, iodoacetamide or p-chloromercuribenzoate) or arginine-specific reagents (2,3-butanedione, 1,2-cyclohexanedione or phenylglyoxal) resulted in a rapid loss of the component B activity. In contrast, component A was resistant to treatment with such reagents, retaining the initial activity almost completely. Farnesyl diphosphate, isopentenyl diphosphate, farnesyl monophosphate and inorganic pyrophosphate protected the synthase against the inactivation by N-ethylmaleimide, farnesyl diphosphate being the most effective. The presence of Mg2+ was essential for the protection by isopentenyl diphosphate and inorganic pyrophosphate. For protection of the synthase activity against the inactivation by 2,3-butanedione, the presence of farnesyl diphosphate, isopentenyl diphosphate and Mg2+ was more effective than that of the individual substrates and Mg2+. Inorganic pyrophosphate provided substantial protection. In the absence of component A, the component B activity was not protected by any substrates or its analogue. These results suggest that the catalytic site of the synthase is formed by cooperative interaction between components A and B, and that cysteine and arginine residues on component B play important roles in the synthase activity.

Aldehydes↗
PubMed2006

Preparation of allyltin reagents grafted on solid support: clean and easily recyclable reagents for allylation of aldehydes.

The preparation of polymer-supported allyltin reagents was shown to be possible for both unfunctionalized and functionalized allyl units. These reagents were treated with aldehydes in the presence of cerium(III) or indium(III) salts to afford high yields of homoallylic alcohols, practically uncontaminated with organotin residues (less than 5 ppm). Some mechanism aspects are briefly discussed and the potential for regeneration and reuse of these supported reagents is pointed out.

Journal Article↗
PubMed1994

Initial formation of a non-covalent enzyme-reagent complex during the inactivation of clostridial glutamate dehydrogenase by Ellman's reagent: determination of the enzyme's dissociation constant for the binary complex with NAD+ from protection studies.

The time-course of reaction between Ellman's reagent (DTNB) and clostridial glutamate dehydrogenase has been investigated over a wide range of reagent concentrations (50-5000 microM) and showed pseudo-first-order kinetics throughout. The reaction was followed both by monitoring loss of enzyme activity and by detection of released thionitrobenzoate through its absorbance at 412 nm, and, when both methods were used for the same DTNB concentration, the pseudo-first-order rate constants were identical within experimental error, suggesting that the two methods detect the same process. The dependence of the rate constants on DTNB concentration clearly shows saturation, with a limiting value of 1.62 x 10(-3) s-1 and a dissociation constant of 1.0 mM governing the formation of the implied non-covalent enzyme-DTNB complex. This information has allowed a detailed analysis of the protection of the enzyme by NAD+, yielding a value of 334 microM for the dissociation constant for the enzyme-coenzyme binary complex. In view of the convenience of protection studies as a means of determining dissociation constants, this study emphasizes the importance of establishing whether a chemical modification reaction follows simple first-order kinetics with respect to the chemical reagent.

Clostridium↗
PubMed1988

Applications of BOP reagent in solid phase synthesis. Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1-29)-NH2.

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.

Growth Hormone-Releasing Hormone↗
PubMed1976

Four commercial urease reagents and a laboratory-prepared reagent compared for analysis of blood urea nitrogen with the Beckman analyzer.

Three laboratory-prepared urease reagents were compared with a commercial preparation supplied for routine use on the Beckman Blood Urea Nitrogen Analyzer. There were discrepancies in results for urea nitrogen among the four urease reagents when matching serum and the corresponding oxalate/fluoride treated plasma were compared as measured with the Beckman Analyzer and continuous-flow (AutoAnalyzer) method. All four urease preparations were affected by fluoride, but to different extents. We believe that an effective laboratory reagent can be prepared in the laboratory at significantly lower cost.

Autoanalysis↗
PubMedDate not supplied

Urinary tricyclic antidepressant screening: comparison of results obtained with Abbott FPIA reagents and Syva EIA reagents.

Human urine was tested for tricyclic antidepressants with fluorescence polarization immunoassay (FPIA) reagents on Abbott's ADx system and with EIA reagents on Syva's ETS system. A 75-ng/mL imipramine calibrator cutoff was used with the ADx system and a 300-ng/mL nortriptyline calibrator cutoff with the ETS system. The ETS system was adapted to analyze tricyclic antidepressant (TCA) samples using the Syva Emittox serum TCA assay. Negative urine was spiked with various tricyclic and tetracyclic antidepressants, phenothiazines, and other medications with potential to interfere with the assays. Verification of samples was performed by thin-layer chromatography and gas chromatography/mass spectrometry. The different compounds were added to urine at concentrations of 200, 400 and 1,000 ng/mL. At 1,000 ng/mL all TCA compounds tested gave positive results with the ADx and ETS systems. However, some non-TCA medications spiked at 1,000 ng/mL gave false positive results with both systems. The tetracyclic antidepressants did not cross-react and gave negative results. Clomipramine-spiked urine at 400 ng/mL yielded a false negative result on the ETS system.

Antibody Specificity↗
PubMed2003

[Influence of reagent evaporation on other laboratory reagents and appropriate countermeasures].

When the automated analyzer was first introduced, abnormally high values of urine creatinine and abnormally low values of serum creatinine were seen. It was shown that evaporation of the reducing agent stabilizer(sulfurous acid), which is present in the serum iron measurement kit, was occurring and that the observed low values of serum creatinine were due to consumption of hydrogen peroxide by gasifying sulfurous acid. After the cause was elucidated, the abnormal lowering of creatinine values was prevented. This experience demonstrates that care must be exercised when introducing new analytical method in clinical laboratories because evaporation of reagents can influence in other reagent systems.

Autoanalysis↗
PubMed1985

Fish eye-lens reagents: a possible new class of reagents for molecular and cellular identification.

Initial studies have shown that fish lens protein can be modified in vitro to acquire a specific affinity for selected molecules, e.g., human hemoglobins, and cells, e.g., human erythrocytes. These studies were extended by preparing lens reagents that can distinguish bovine serum albumin from ovalbumin, and major groups of human erythrocytes in the ABO system from each other. Lens reagents appear to be potentially useful tools for the identification of specific molecules, either in solution or on cell surfaces.

Agglutination Tests↗
PubMed1998

"Propylene spaced" allyl tin reagents: a new class of fluorous tin reagents for allylations under radical and metal-catalyzed conditions.

A new generation of propylene-spaced fluorous allyltin reagents [(Rf(CH2)3)3SnCH2CH = CH2] is described. These succeed in radical allylations where their lower homologs (ethylene-spaced) fail, and they provide improved performance in transition metal catalyzed allylations. The reagents and byproducts are readily separated by simple fluorous-organic liquid-liquid or solid-liquid extractions.

Aldehydes↗
PubMed1989

Guidelines for evaluation of reagent strips. Exemplified by analysis of urine albumin and glucose concentration using visually read reagent strips. International Federation of Clinical Chemistry, Scientific Division.

Reagent strips are frequently used in the practice of laboratory medicine as well as outside the professional laboratories. In the present report, a scheme is presented which allows a reproducible description of the performance of reagent strips. This is exemplified using strips intended for estimation of urinary albumin and glucose. Experimental conditions that are particularly important for these components are given. Suitable and necessary items which should be included in the protocol presenting results of an evaluation are listed. The guidelines might have general applicability in evaluation and description of tests with a limited number of possible results.

Albuminuria↗
PubMed1985

Clinical utility of a monoclonal direct fluorescent reagent specific for Legionella pneumophila: comparative study with other reagents.

Twenty-four lower respiratory tract samples taken from patients with culture-confirmed Legionella pneumophila infection were examined with three different direct immunofluorescent antisera to L. pneumophila, as were 29 samples from similar sources taken from patients without Legionnaires disease. The reagents studied were Genetic Systems Corp. (GS) monoclonal L. pneumophila conjugate, which reacts with all known serogroups of L. pneumophila, BioDx polyvalent L. pneumophila serogroups 1 through 6 conjugate, and Centers for Disease Control polyvalent pool A L. pneumophila serogroups 1 through 4 conjugate. The specimens had been frozen at -70 degrees C for 0.5 to 5 years. Randomization was used in coding the samples, which were stained and read by an independent observer. All three conjugates correctly identified all positive and negative samples. No difference was noted among the conjugates in the absolute numbers of fluorescent L. pneumophila bacteria per sample. The GS conjugate had a much cleaner background than did the other two reagents. Mean staining intensity scores were 3.4, 3.9, and 3.7 for the GS, BioDx, and Centers for Disease Control conjugates, respectively. This study demonstrates that the diagnostic efficiency of all three conjugates is equivalent. Since the GS conjugate is easier to read, does not cross-react with non-L. pneumophila bacteria, and reacts with serogroups 1 through 10 of L. pneumophila, it appears to be preferable for use in diagnostic testing on nonhistopathologically processed specimens.

Antibodies, Bacterial↗
PubMed1993

Labeling of cysteine 231 in acetylcholinesterase from Torpedo nobiliana by the active-site directed reagent, 1-bromo-2-[14C] pinacolone. Effects of 2,2'-dipyridyl disulfide and other sulfhydryl reagents.

Acetylcholinesterase (AcChE, EC 3.1.1.7) was isolated from the electric organ of T. nobiliana and treated with the active-site-directed alkylating agent 1-bromo-2-[14C]pinacolone ([14C]BrPin), or with BrPin, which acts initially as a competitive inhibitor, Ki = 0.18 mM, and then inactivates the enzyme, k2 = 1.8 x 10(-4) s-1. AcChE aliquots were digested with trypsin and fractionated by reversed phase high performance liquid chromatography. Inactivation caused a decrease in one absorption peak and an increase in another, identified as the peptide beginning at Ala-222 and extending to Arg-242. 5-Trimethylammonio-2-pentanone, a competitive inhibitor, isosteric with acetylcholine, retarded the inactivation and decreased the quantity of labeled peptide. On sequencing, the 14C label was found associated with Cys-231. This was confirmed by comparison with synthesized S-pinacolonylcysteine, by study of effects of blocking the sequencing by o-phthalaldehyde, and by inactivation by 2,2'-dipyridyl disulfide (2-PDS), a thiol-specific reagent that acts initially as a competitive inhibitor, Ki = 0.042 mM, and then inactivates the enzyme, k2 = 5.0 x 10(-4) s-1. This is retarded by 5-trimethylammonio-2-pentanone, and prior inactivation by 2-PDS prevents subsequent reaction of [14C]BrPin in the active site. BrPin inactivates AcChEs from Electrophorus electricus and from human erythrocyte, but 2-PDS does not. Neither reagent inactivates butyrylcholinesterases from human and horse serum.

2,2'-Dipyridyl↗
PubMed1994

Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents.

The need for the standardization of reagents and methods used in the histology laboratory is demonstrated. After definitions of dyes, stains, and chromogenic reagents, existing standards and standards organizations are discussed. This is followed by practical instructions on how to standardize dyes and stains through the preparation of reference materials and the development of chromatographic methods. An overview is presented of the problems concerned with standardization of the Romanowsky-Giemsa stain for cytological and histological application. Finally, the problem of how to convince routine dye and stain users of the need for standardization in their histology laboratories is discussed.

Animals↗
PubMed1974

Inhibition of enzymes by metal ion-chelating reagents. The action of copper-chelating reagents on diamine oxidase.

1. The inhibition of diamine oxidase has been studied by using the following copper-chelating reagents: 1,10-phenanthroline; 2,2'-bipyridyl; 8-hydroxyquinoline (oxine); diethyldithiocarbamate and dithio-oxamide (rubeanic acid). 2. Addition of chelating reagent caused a rapid inhibition of enzyme to a degree dependent solely on the final inhibitor concentration. Addition of substrate gave linear initial rates of reaction showing that under these conditions the inhibition was not being rapidly reversed. 3. The inhibition has been investigated by using new graphical methods and has been found in all cases to involve the chelating agents completely removing two Cu(2+) ions from the enzyme. An alternative possibility, involving ligand substitution, was eliminated. 4. A value of K=8.0x10(-33)m(-2) has been found for the enzyme in equilibrium with 2 Cu(2+) ions (i.e. beta(2), the stability constant for diamine oxidase/two Cu(2+), is 32.1).

Amine Oxidase (Copper-Containing)↗
PubMed1977

The van urk-Salkowski reagent--a sensitive and specific chromogenic reagent for silica gel thin-layer chromatographic detection and identification of indole derivatives.

The chromogenic reagent described has been tested with seventy-nine indole derivatives and found to be very sensitive and indole-specific. The lower limit of detection on silica gel thin-layer plates was between 25 and 50 ng for most indoles. Phenols and hydroxy-, and amino-benzoic acids, hydroxy-, and methoxy-cinnamic acids did not yield chromophores with the exception of p-amino-benzoic acid and p-hydroxy-cinnamic acid which gave yellow and pink chromophores at concentrations greater than 1 and 2 mug. Although many of the C-3 substituted indoles such as indole-3-acetic acid and tryptamine had colors in the reddish-violet-blue color region, most exhibited sufficient color differentiation to allow their identification by thin-layer chromatography. The procedure was simple and required only 10 min from the time of spraying the thin-layer plate until full color development was reached. The colors had a wide spectral range from yellow of the indole-3-glyoxylamide chromophore to blue of the melatonin chromophore, and were extremely stable.

Benzaldehydes↗
PubMed1983

Modulation of optical density by sulfhydryl reagents in microbiuret method: a modified method for protein determination in the presence of sulfhydryl reagents.

2-Mercaptoethanol increases the optical density of assay solutions at wavelengths between 280 to 400 nm, and therefore interferes with the measurement of protein concentration by the microbiuret method. Protein concentration can be determined in the presence of 2-mercaptoethanol up to 6 mM by modification of the method as follows: after the precipitation of protein by trichloroacetic acid in the presence of deoxycholate, the precipitate is resolubilized with NaOH solution. Dithiothreitol interfered with the protein determinations could by made in the presence of 4 mM of dithiothreitol with the modified microbiuret method. This modified method is time-saving and more reliable than other methods for protein determination, such as Lowry's method, in the presence of sulfhydryl reagents.

Proteins↗
PubMed1981

Cell-mediated lympholysis responses against autologous cells modified with haptenic sulfhydryl reagents. II. Analysis of the genetic control and H-2-restricted pattern of cytotoxic responses to sulfhydryl and amino reactive reagents.

N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylene-diamine modified autologous cells are shown to induce respective high and low AED-self CTL responses by spleen cells from H-2b and H-2k mice. The lysis mediated by effector populations from both haplotypes was highly H-2 restricted. Cytotoxic responses induced by a 2nd sulfhydryl- (SH) specific reagent, dithionitrobenzoic acid (DTNB), were shown not to be cross-reactive at the effector cell level with AED-self but exhibited the same high and low CTL response patterns as those induced against AED-self. These Ir-effects reported for the 2 SH reactive compounds are discussed in relation to the opposite Ir-patterns demonstrated against a number of amino (NH2) reactive compounds. The difference of self-recognition sites is suggested to account for the contrasting results observed in the response patterns induced by SH- and NH2-reactive compounds. The AED-self CTL response was found to exhibit Kk-self over Db-self Ir regulation. Thus, this latter type of genetic control appears to function independently of whether the hapten is an SH- or NH2-reactive compound.

Animals↗