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PubMed1995

Determination of sulfamethazine residue in chicken serum and egg by high-performance liquid chromatography with chemiluminescence detection.

A sensitive method that involves high-performance liquid chromatography with chemiluminescence detection is developed for the determination of sulfamethazine residue in samples of chicken serum and egg. Sulfamethazine is extracted from the samples and derivatized with fluorescamine. The derivatized samples are eluted by reversed-phase chromatography using a mixture of 10 mM potassium phosphate and 30% acetonitrile on a Nova-Pak C18 column. The post-column reagents in the chemiluminescence system are 1 mM bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl] oxalate and 0.3M hydrogen peroxide in acetonitrile. The detection limit in the standard solution is 1 ng/mL, and the calibration curve is linear between 1 and 100 ng/mL. The recoveries of spiked samples (50 ppb) are 95.8 +/- 9.7% in chicken serum samples and 84.9 +/- 10.7% in egg samples. In actual sample analysis, the lowest detectable concentration of sulfamethazine (0.073 microgram/mL) was found at 48 h in serum and on day 9 (0.017 microgram/g) in egg after oral administration of a dose of 100 mg/kg body weight to hens.

Animals↗
PubMed1983

The effect of malonyldialdehyde, a product of lipid peroxidation, on the deformability, dehydration and 51Cr-survival of erythrocytes.

Erythrocyte membrane lipid peroxidation has been reported to occur in various haemolytic anaemias. In the present study, treatment of human erythrocytes with malonyldialdehyde (MDA), a product of fatty acid peroxidation, induced membrane rigidity, cellular dehydration and reduced whole cell deformability. These effects of MDA were blocked by histamine and fluorescamine, which can act as alternate substrates for MDA. Additionally, reduced deformability of MDA-treated rabbit cells was associated with shortened 51Cr survival in vivo. These findings suggest a biochemical basis for decreased survival of erythrocytes undergoing peroxidative damage of the membrane.

Animals↗
PubMed2006

Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation.

The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.

Animals↗
PubMed1979

Reaction of formaldehyde with calf-thymus nucleohistone.

The reactions of formaldehyde with calf thymus nucleohistone were analyzed in the following ways: measurement with fluorescamine of the decrease in primary amino groups resulting from hydroxymethylation and crosslinking reactions, measurement with dodecylsulphate-gel electrophoresis of formation of histone oligomers, measurement of fixation of histones to the DNA in nucleohistone, and measurement of changes in the circular dichroism spectrum in the region of 250--300 nm. In the presence of formaldehyde, the primary amino groups of histones decreased very rapidly, attaining an equilibrium within 60 min, and successively intermolecular crosslinks were also formed between histone molecules, the resulting dimers and oligomers being separable by dodecylsulfate-gel electrophoresis. Whereas the fixation reaction proceeded much more slowly. The extent of fixation could be measured more accurately by dodecylsulfate/sucrose centrifugation analysis than by sulfuric acid extraction. After removal of formaldehyde from the reaction mixture, the fraction of masked amino groups decreased, perhaps due to the reverse reaction, but the extent of fixation of histones continued to increase with time. No specificity was observed among five molecular species of histones in the fixation reaction. With increase in formaldehyde concentration, the ellipticity of nucleohistone decreased to a minimum with about 0.4% formaldehyde, and then increased.

Amino Acids↗
PubMed1981

Activity of phospholipase A2 in the inner membrane of rat-liver mitochondria.

The inner membrane of rat liver mitochondria contains a highly active phospholipase A2 which has alkaline pH optimum and requires Ca2+ in the micromolar range. The phospholipase is particularly active on the endogenous phosphatidylethanolamine and release relatively high amounts of docosahexanoic acid. The phospholipase A2 of mitochondria or mitoplasts is not dependent on calmodulin. Using fluorescamine-labelled mitoplasts there are indications that the enzyme is localized on both sides of the inner membrane.

Animals↗
PubMedDate not supplied

Molecular structure of human seminal coagulum: the role of proteolysis.

The techniques of SDS-polyacrylamide slab gel electrophoresis and a sensitive fluorescamine-based method were used to study autoproteolysis of human seminal coagulum at various pH. The major protein bands of 72 and 55 Kd were optimally degraded at pH 7.5. Degradation at pH 3.5 was preceded by a lag period of 30 min. When the seminal coagulum was incubated for 5 min at pH 7.5 and then adjusted to pH 3.5, degradation was more rapid and complete than at pH 3.5 only. Autoproteolytic degradation product of discreted sizes were obtained when compared with degradation in the presence of pronase.

Adolescent↗
PubMedDate not supplied

Fluorometric determination of acid proteinase activity in vulvovaginal candidosis.

Vulvovaginal candidosis is one of the most frequent disorders in obstetrics and gynaecology. Candida albicans is commonly considered to be the true vaginopathic agent. The secreted acid proteinase might be especially relevant in the pathogenesis of vulvovaginal candidosis. A fluorometric determination of acid proteinase activity of clinical C. albicans isolates was carried out during the present work using fluorescamine. L-Leucyl-L-alanine was included as an internal standard and the results were expressed as nmoles of leucylalanine equivalents h-1 per 2 x 10(4) cells. The 13 isolates were taken from non-diabetic, non-pregnant women aged 22-35 years with vulvovaginal candidosis. Candida albicans ATCC 44858 was used as a control. The proteinase activity in culture supernatants was detectable starting from the mid- to late- exponential phase of growth, peaked between 30 and 46 h, and then declined. The control had an activity of 2.72 nmol h-1 per 2 x 10(4) cells, whereas eight of the samples had a lower activity (1.05 nmol h-1 per 2 x 10(4) cells on average) and five of the samples had a higher activity (6.53 nmol h-1 per 2 x 10(4) cells on average). The fluorometric determination of acid proteinase activity was found to be more reproducible and sensitive than the previously used spectrophotometric determinations.

Adult↗
PubMed1991

Fluram induces species-dependent C and G bands in mammalian chromosomes, revealing heterogeneous distribution of chromosomal proteins.

Fluram (Fluorescamine; 4-phenylspiro(furan-2(3H),1'-phthalan)-3,3'-dione) is a fluorogenic reagent, which permits the detection of primary amines by forming highly fluorescent pyrrolinone derivatives. This reagent has been used on methanol-acetic acid fixed metaphase chromosomes of mouse and man and proved to be very effective in differentiating chromosome regions in both genomes. Mouse centromeric heterochromatin is highly reactive, showing intense fluorescence in all centromeric regions, whereas human chromosomes show no fluorescence in such regions. In addition, a G-like banding pattern is also obtained in both types of chromosomes. The differential reactivity of each chromosome region showed by this method demonstrates a heterogeneous distribution of chromosome proteins, resulting in a chromosome banding pattern, which is in this case species dependent.

Animals↗
PubMed1979

The effect of sulfhydryl and amino group reagents on human lymphocyte--sheep erythrocyte rosettes.

We have studied the influence of certain chemical groups, located on the surface of lymphocytes or sheep red blood cells, on the ability of these cells to form spontaneous rosettes. We found that fluorescamine, which reacts with amino groups, inhibits rosette formation, while p-chloromercuriphenylsulfonic acid, which binds to sulfydryl groups, increases the percentage of the rosette-forming lymphocytes. The possible mechanism of action of the above reagents is discussed.

4-Chloromercuribenzenesulfonate↗
PubMed1997

[Methodological approach to regulation of polyamine].

This brief article is dedicated to the late Professor Morizo Ishidate, and concerned in a methodology developed by the author and collaborators, aiming at the regulation of polyamine, especially, the regulation of spermidine synthase. The content is separated in 3 sections. The first section on the development of analytical methods, contains seven items, e.g. fluorometric determination of polyamines by fluorescamine, analysis of naturally occurring polyamines by GC and GC-MS, etc. The second section on the syntheses of needed compounds, contains three items, e.g. syntheses of decarboxylated S-adenosylmethionine which is a substrate for spermidine synthase and its related compounds, syntheses of 15N-enriched polyamines applicable to the preparation of various polyamines, etc. The last section on the studies of aminopropyl transferases, contains three items, e.g. purification of spermidine synthase from mammalian tissues using ATPA-Sepharose, a novel affinity carrier, newly developed inhibitors for the enzymes, etc.

Animals↗
PubMed1976

A simple and sensitive fluorometric assay method for taurine using high-voltage paper electrophoresis.

A simple and sensitive fluorometric assay method for taurine (2-aminoethanesulfonic acid) has been developed. For the separation of taurine, high voltage paper electrophoresis subsequent to column chromatographic procedures was employed. Fluorescent product of taurine was yielded by spraying fluorescamine (4-phenylspiro [furan-2(3H), 1'-phthalan]-3, 3'-dione) and borate buffer on the paper, and the fluorescence was assayed spectro-fluorometrically after eluting with 50% ethanol. The linear relationship between the concentration of taurine and fluorescence developed was achieved over the concentration ranges of 0.5-10 nmoles, and the recoveries obtained were 90-100%. The specificity of this method for taurine was satisfactory and structural analogues involved in the metabolic pathway of taurine did not interfere with the assay. Examples for tissue levels of taurine in various organs of the rat as determined by this new method are also presented.

Animals↗
PubMed1977

A rapid fluorometric assay of angiotensin-converting enzyme.

A rapid, simple and sensitive method is described for the fluorometric assay of angiotesin-converting enzyme using Fluorescamine. The critical factors such as optimal pH, incubation time, chloride ion, and inactivation by EDTA and 8-hydroxyquinoline were examined. The Km value for hippuryl-L-histidyl-L-leucine was 0.5 mM. This method was applied to the assay of angiotensin-converting enzyme in the rat serum and the reproducible values were obtained with a 10 mul of the rat serum.

Animals↗
PubMed1988

Alterations in organization of phospholipids in erythrocytes as factor in adherence to endothelial cells in diabetes mellitus.

Erythrocytes from patients with diabetes mellitus exhibit increased adherence to cultured human vascular endothelial cells. We investigated the alterations in erythrocyte surface characteristics that may contribute to their abnormal adherence. The organization of phospholipids in the lipid bilayer, as determined by phospholipase A2 treatment and chemical labeling with fluorescamine and trinitrobenzene sulfonic acid (TNBS), is altered in erythrocytes from diabetic patients. Specifically, 12-18% of phosphatidylserine in diabetic erythrocytes (n = 25) is accessible to phospholipase A2 hydrolysis and TNBS labeling, compared to none in normal subjects. These results suggest either a loss in lipid asymmetry or in vivo destabilization of erythrocyte membranes in diabetic patients, causing increased accessibility to phospholipase A2 degradation. The dye merocyanine 540 (MC-540), which is sensitive to the packing of lipids in the bilayer of the membrane, revealed more binding and fluorescence in erythrocytes from diabetic patients than in those from normal subjects. On flow cytometric analysis, 64.5 +/- 17.0% red blood cells (RBCs) in diabetic patients, compared to 35.1 +/- 25.9% RBCs in normal subjects, showed positive MC-540 binding, indicating significant (P less than .001) differences in the packing of lipids in the external leaflet of the bilayer. The results of our study suggest that a loss of lipid asymmetry and/or less ordered packing in the outer leaflet of the diabetic erythrocyte membrane may be responsible for the increased propensity of erythrocytes to adhere to vascular endothelium.

Adult↗
PubMed1975

Fast, short-column separation and fluorometric determination of monosodium glutamate in foods.

In a simple and fast procedure, monosodium glutamate in food products is first separated by a short ion exchange column and subsequently determined fluorometrically with fluorescamine. An average recovery of 90.8% with a standard deviation of plus or minus 6.67% was obtained for added monosodium glutamate in a series of 8 products. As little as 0.05% monosodium glutamate can be determined. The method is faster than the official method and saves 5 hr/determination.

Chromatography, Ion Exchange↗
PubMed2002

[Determination of 7-aminoflunitrazepam, the major metabolite of flunitrazepam in urine by high performance thin-layer chromatography].

A rapid and highly sensitive method, based on high performance thin-layer chromatography, is described for the qualitative and semiquantitative determination of 7-aminoflunitrazepam, the main metabolite of flunitrazepam in human urine. 7-Aminoflunitrazepam in specimen was extracted by solid phase extraction using GDX-403 porous polymer bead as sorbent and ethyl ether as eluant. Fluorescamine was used as a reagent to produce fluorescent product on the plate. The limit of detection was 5 micrograms/L and the limit of quantitation was 15 micrograms/L for 7-aminoflunitrazepam in urine. The method can be successfully used for measuring 7-aminoflunitrazepam in urine samples of the subjects excreted over a 48 h period after receiving 1 mg flunitrazepam orally. The method is applicable to drug examination for the cases of drug-facilitated robbery and rape.

Chromatography, Thin Layer↗
PubMed1989

Characterization of chemical modifications of surface properties of low density lipoproteins.

Low density lipoproteins (LDL) were modified by incubation with formaldehyde and malondialdehyde and by autoxidation. Different methods were developed to measure alterations of surface properties of LDL. A method based on fluorescamine fluorescence was used to measure changes of free amino groups. All modified LDL samples have a decreased number of amino groups. As shown by gel electrophoresis, the negative surface charge of LDL increases. Aggregation of LDL induced by poly(ethylene glycol) (PEG) is influenced by LDL modification. The PEG 6000 concentration necessary for LDL aggregation is shifted to higher values for modified LDL due to the increased negative surface charge. This result may be of interest for the application of PEG for selective separation of different classes of lipoproteins. The formation of aggregates of LDL induced by dextran sulphate is reduced due to the loss of positive charges of amino groups on the LDL surface.

Dextran Sulfate↗
PubMed1987

Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site.

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.

Amino Acid Sequence↗
PubMed1979

A fluorescence energy transfer system for studying the functional properties of chromatin.

3YI-B-1 rat embryo fibroblasts were used for double fluorescent labeling. The labeling was carried out with fluorescamine and ethidium bromide as an energy donor-acceptor pair. The fluorimetric measurements proved the existence of an energy transfer process. The parameters of this transfer depended on both cell and dye concentrations. It is this character that makes this labeling technique promising as a means of distinction between cells with different structures.

Animals↗