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PubMed1983

Fluorometric assay of kininase activities in rat tissues.

A simple method for the assay of bradykinin (BK)-degrading enzymes was investigated. The procedure of the method includes enzymatic degradation of BK, separation of the residual BK on a small P-cellulose column (0.6 X 3 cm), and its fluorometrical determination based on the reaction with fluorescamine. BK was separated completely from its fragments produced during enzymatic reaction by the column chromatography. The recovery rate of BK was 96 +/- 3%. Quantitative determinations could be carried out on 0.2 nmol of BK, at least in the fluorometry. This method was available for the assay of the enzymes in tissue homogenates as well as in purified preparations, and its usefulness for the study of the enzymes is presented.

Animals↗

PubMed1984

High-performance liquid chromatographic analysis of ammonium in human urine.

A method for the quantitative determination of ammonium in human urine by high-performance liquid chromatography (HPLC) is described. After making fluorescent substances with fluorescamine, they were separated and quantified by their fluorometric intensity. The intensity (as measured by peak height) was linear between 0.5 and 5.0 micrograms, and coefficients of variation for elution time and peak height on replicate analysis of the standard were 0.15 and 4.2%, respectively. Recoveries of added ammonium were 96.5 and 97.3%, respectively, on 2.0 and 3.0 micrograms by this method. Detection limit of this method was 0.2 microgram. There was good agreement between the proposed HPLC method (X) and ion chromatographic method (Y), giving the relationships as Y = 0.956X + 0.012, r = 0.991.

Adult↗

PubMed1984

Use of o-phthalaldehyde to reduce background during automated Edman degradation.

Background generated during the cleavage step of the Edman degradation is a major obstacle to extended automated amino acid sequencing. It was demonstrated recently by A. S. Bhown, J. C. Bennett, P. H. Morgan, and J. E. Mole (1981, Anal. Biochem. 112, 158-162) that introduction of fluorescamine into the spinning cup reduced background by blocking primary amino groups at cycles where a proline residue is at the exposed amino terminus. A convenient blocking reaction program using o-phthalaldehyde which can be intercalated into the sequencing format of an automated Beckman liquid-phase sequencer is presented. The advantages of o-phthalaldehyde in blocking of primary amines when proline is amino terminal arise from its stability in aqueous solution and the ease of programmed metering of delivery to the sequencer spinning cup. The blocking reaction proved successful not only in extending sequence analyses but also in the elimination of unwanted sequences in selected peptide mixtures without the necessity of purification of the target peptide.

Aldehydes↗

PubMed1987

Location and purification of enzymes on polyacrylamide gels using dialyzable fluorescent markers.

A method for the location of proteins/enzymes by polyacrylamide gel electrophoresis using dialyzable low-molecular-weight fluorescent peptide markers is described. The markers prepared by treating the peptic digest of casein with fluorescamine showed several bands on gel electrophoresis which helped in locating the proteins. The located desired protein could subsequently be purified by extraction.

Aldose-Ketose Isomerases↗

PubMed1989

Reductive methods for isotopic labeling of antibiotics.

Methods for the reductive methylation of the amino groups of eight different antibiotics using 3HCOH or H14COH are presented. The reductive labeling of an additional seven antibiotics by NaB3H4 is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented.

Aminoglycosides↗

PubMed1979

Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis.

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of protein solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.

Blood Platelets↗

PubMed1990

Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes.

Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50 degrees C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability under low ionic conditions, and the binding of spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluorescence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than that observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably increased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. In spite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.

Actins↗

PubMed1983

Native molecular weight of adenovirus proteins: on the oligomeric structure of the fiber.

Fluorescamine-modification of amino groups was used to eliminate the influence of basic charge on the final migration position of protein's in alkaline pH polyacrylamide gradient gel electrophoresis. As applied to adenovirus structural components, this type of analysis suggested the fiber to be composed of three identical subunits. The trimeric nature of both penton base and fiber therefore displaces the problem of symmetry mismatching to penton base and surrounding hexons at each vertex of the adenovirus icosahedron.

Adenoviridae↗

PubMed1987

The effect of lithium on the membrane molecular dynamics of normal human erythrocytes.

Erythrocytes from normal adults with no personal or family history of bipolar affective disorder were analyzed by fluorescence spectroscopy to determine what effect, if any, acute in vitro incubation with lithium had on erythrocyte membrane dynamics. The effects on erythrocyte membrane molecular dynamics of varying concentrations of Li2CO3 (0.25-2.0 meq/liter), varying incubation temperatures (25-40 degrees C), and varying incubation times (5-185 min) were investigated. Following incubation with Li2CO3, the erythrocytes were labeled with either 4-phenylspiro-[furan-2(3H),--1'phthalan]--3,3'-dione (fluorescamine), which binds to membrane surface primary amines, or 12(9)anthroyl stearate [12(9)AS], which inserts deep in the membrane hydrocarbon core. The membrane molecular dynamics were then determined by fluorescence anisotropy measurements. These studies demonstrate that clinically relevant concentrations of Li+ incubated with intact normal human erythrocytes significantly alters molecular dynamics on the erythrocyte membrane surface, with less striking changes in the hydrocarbon core. A possible interpretation of these findings is that hydrated Li+ alters the electrostatic interaction of membrane surface molecules, as well as the surrounding solvent (water) structure, with a resultant increase in the molecular motion of these molecules. Alterations in membrane receptor motion could potentially alter receptor functional activity. If similar motional alterations were to occur in the interior of a membrane channel, such as an ionophore, the functional activity of the channel could also be potentially altered. These findings provide additional insight into possible biological actions of Li+, as well as potential molecular alterations in bipolar affective disorder erythrocytes.

Adult↗

PubMed1982

Determination of endotoxin using fluorescent probe.

Bacterial endotoxin induces gel-formation of amebocyte lysate. This gelation test, the limulus test, was first described by Levin and Bang [1]. The limulus test has been widely used for the detection of endotoxin, not only in fundamental bacteriological research but also in clinical diseases [2]. Although the technique of the limulus test is simple, the endotoxin level can not be determined quantitatively. We developed a method to determine the endotoxin in physiological saline solution and glucose solutions using a fluorescent probe, fluorescamine.

Endotoxins↗

PubMed1991

Enzymatic fluorometric assay for plasma pyridoxal 5'-phosphate.

An enzymatic fluorometric assay to quantitate plasma pyridoxal 5'-phosphate (PLP) is described. PLP is preincubated for 30 min with purified tyrosine decarboxylase apoenzyme (TDA) in acetate buffer and is then incubated with L-tyrosine for 60 min. The decarboxylated metabolite, tyramine, is extracted into ethyl acetate, air dried, dissolved in borate buffer and reacted with fluorescamine to form a fluorophor (395/475 nm). Recovery of exogenous PLP was 83%. After log-normal transformation of 129 plasma PLP levels obtained from 3 groups of subjects (in-patients, out-patients, staff), a normal distribution curve was obtained yielding a mean of 83 nmol/L. With 95% confidence level, the reference limits were 32-170 nmol/L. There were no age or sex differences in mean plasma PLP levels between any of the 3 groups of subjects studied.

Adolescent↗

PubMed1990

Capillary electrophoretic separations of peptides using micelle-forming compounds and cyclodextrins as additives.

The value of electrokinetic capillary chromatography for separating structurally similar model peptides and tryptic digests is demonstrated. The behavior of model peptides in buffer systems containing dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, sodium dodecyl sulfate and two cyclodextrins as additives is described. These additives, under different analytical circumstances, exhibit certain beneficial effects for peptides with similar net charges but different hydrophobicities. Separations of underivatized peptides, utilizing UV detection, are presented. In addition, separation of fluorescent products of peptides derivatized with o-phthalaldehyde, fluorescamine, and a new reagent, 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde, are demonstrated and discussed. Beneficial spectroscopic detection effects with cyclodextrin are also noted.

Chromatography↗

PubMed1992

Effect of buffer constituents on the determination of therapeutic proteins by capillary electrophoresis.

Capillary electrophoresis has proved to be a versatile method for the determination of proteins, peptides and amino acids in pharmaceutical formulations. For quantification of the capillary electrophoresis data, however, significant errors may result if the analysis is performed using improper separation conditions. The peak area response for protein analytes, which is generally low in conventional UV detection, may also vary dramatically depending on the nature of the buffer used in the separation. This paper describes the effects of various buffer constituents and analytical conditions on the capillary electrophoretic separation and quantification of a humanized monoclonal antibody in bulk form and in a typical therapeutic formulation. For optimum peak area response and reproducibility, protein derivatization with an appropriate chromophore (e.g., fluorescamine) and separation in the presence of a moderate ionic strength buffer containing lithium chloride, tetramethylammonium chloride or trimethylammonium propylsulfonate is recommended. General guidelines for the determination of proteins by capillary electrophoresis and a rationale for the use of internal standards to improve the quantification of data are also discussed.

Antibodies, Monoclonal↗

PubMed1979

Fluorescent labelling of proteins of lymphocyte plasma membranes.

Fluorescamine has been used for labelling proteins present on the surface of normal human peripheral blood lymphocytes. Under the conditions of study, 12 labelled proteins could be detected by SDS gel electrophoresis. This method may be of value in biochemical studies of lymphocyte membranes.

Antigens, Surface↗

PubMed1979

A spectrofluorometric protein assay of plaque on dentures and of denture cleaning efficacy.

A spectrofluorometric method of protein analysis using fluorescamine was shown to be a meaningful method of evaluating denture cleanser efficacy. This assay was useful in the presence of most currently used denture cleanser ingredients, materials which cause other protein assay methods to fail. The determination of dental plaque protein was shown to be an adequate measure of dental plaque dry weight.

Borates↗

PubMed1987

Collagenolytic activity in the human leukemic K-562 cell line before and after induction of erythroid differentiation.

K-562 cells were studied for their ability to produce collagenolytic enzymes in culture. Collagenolytic activity has been assayed by a fluorescamine method, it requires Ca2+ ions and is optimal at pH 7.5. It is present within the cells in the cytoplasm and bound to membranes, in addition it is excreted into the medium. Cellular collagenolytic activity is activated by trypsin and inhibited by cysteine and serum. Total cellular activity of K-562 cells corresponds to 30 and 60% of that found respectively in human granulocytes and promyelocytic human HL 60 cells. Induction by hemin of erythroid differentiation in K-562 cells leads to a reversible decrease in collagenolytic activity which is concomitant with the recruitment of hemoglobin-synthesizing cells. The use of spontaneously low and highly hemoglobinized clones of K-562 cells confirms that collagenolytic activity is inversely correlated with the hemoglobin content.

Blood↗

PubMed1981

Separation of cell subpopulations from embryonic chick neural retina with fluorescence-activated cell sorting.

Fluorescence-activated cell analysis and sorting was used to characterize and separate cell subpopulations from the developing chick retina according to size, stage in the cell cycle and location in the tissue. It was shown that uptake of rhodamine B isothiocyanate (RITC) protein stain, light-scattering at large angles, and the time of flight of cells through the laser beam all correlate with cell size. Vital staining of DNA by H33342 permits cell separation according to phase in the cell cycle (G1, S, G2 + M). Further, a method was developed for separating postmitotic cells, based upon the quenching of H33342 fluorescence by the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA. Quantitative data reflect the decrease of DNA-synthesizing cells and the increase of postmitotic cells as the development of the retina proceeds. Isolation of cells on the basis of their location in the whole tissue is achieved by exposing the desired surface (e.g. the inner (concave) surface of the retina, which is rich in ganglion cells) to a suspension of Fluorescamine, which stains only cell layers close to the surface. Sorting out the fluorescing cells yields a high proportion of cells with the appearance, size and postmitotic characteristics of ganglion cells.

Animals↗

PubMed1989

Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus.

Isopenicillin N epimerase, which catalyzes conversion of isopenicillin N to penicillin N, has been purified to electrophoretic homogeneity from the cell-free extract of Streptomyces clavuligerus by a procedure involving ammonium sulfate fractionation and chromatographies with DE-52, DEAE Affi-gel blue, Sephadex G-200, calcium phosphate-cellulose, and Mono Q. The purified epimerase is monomeric with a molecular weight of 47,000 or 50,000 as estimated by SDS-polyacrylamide gel electrophoresis or gel filtration, respectively. The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of protein, and shows absorption maxima at 280 and 420 nm. The epimerase catalyzes the complete 'racemization' on both the L-alpha-aminoadipyl side-chain of isopenicillin N and the D-alpha-aminoadipyl side-chain of penicillin N, so that an approximately equimolar mixture of the two penicillins is produced. The mixture is not truly racemic, since these penicillins are diastereomers rather than optical isomers. The chemical modification of primary amino groups of the epimerase by fluorescamine results in a great loss of the enzyme activity. The activity of purified enzyme is partially stimulated by the addition of sulfhydryl compounds. The activity is strongly inhibited by sulfhydryl group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.

Amino Acid Isomerases↗