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At least 19 recordsLinked to original sources
PubMed1976

Theory of fluorescence polarization of oriented pigment molecules in spherical arrays.

Theoretical results are presented which are appropriate for the analysis of the static polarized fluorescence experiment with oriented pigment molecules in spherical arrays (vesicles). Though the global orientation mediated over the whole sphere is isotropic, the fluorescent molecules may have preferred local orientation with respect to the local plane. As in a former paper, concerning fluroescence polarization in planar arrays, three basic (local) orientation distributions of the electronic transition moments are investigated, which may be expected to describe a wide class of real cases with sufficient accuracy. Analytic expressions for the degree of polarization are derived. One important result is that the degree of polarization may be extremely dependent on the local orientation of transition moments. Hence the usual method of determination of microviscosities from experiments with vesicles with the use of the theory of fluorescence polarization for macromolecules in solutions should be regarded with great caution.

Electromagnetic Phenomena↗
PubMed1979

Assay of angiotensin I by fluorescence polarization method.

Fluorescence polarization technique was applied for the assay of angiotensin I (Al) in human plasma. In this assay system, fluorescein labeled Al (F-Al), which retained the original antigenicity, and antibody to Al was allowed to interact in a cuvette in the instruments yielding an increase in the fluorescence polarization (P) value. Non-labeled Al in the sample blocked the binding of F-Al to the antibody resulting lower P value. Log of antigen concentration and P value was found to exhibit reverse linear proportionality between 0.05 ng to 2 ng/ml of antigen (Al) concentration. The present method was compared with standard radioimmunoassay method and the result showed that data were compatible with each other. The calculation of P value is automated and three cavity filter and optics of the instrument gave reliable results. The method is fast (less than 2 min), sensitive (less than 10 picomole/ml) and simple (no separation step before readout of the results).

Angiotensin I↗
PubMed1979

Direct fluorescence polarization immunoassay.

Direct fluorescence polarization immunoassay of serum cortisol was established. Non-specific binding of fluorescent-labelled cortisol to serum proteins was successively eliminated by sodium dodecyl sulfate. The minimal amount of cortisol detected was 0.1 ng/tube and serum concentration of 1.0 microgram/dl to 100 microgram/dl of cortisol could be measured. This fluorescence polarization immunoassay satisfied the standard criteria of accuracy and precision. The values correlated well with those obtained by radioimmunoassay.

Fluorescent Antibody Technique↗
PubMed1978

Assay of an antitumor protein, neocarzinostatin, and its antibody by fluorescence polarization.

I evaluated use of the fluorescence polarization technique to measure neocarzinostatin, a proteinaceous antitumor antibiotic, and its antibody, in serum. The antigen (neocarzinostatin), labeled with fluorescein isothiocyanate, was allowed to interact with its antibody in a cuvet, in the instrument, yielding an increase in the fluorescence polarization value. Antibody content was determined in the presence of a definite amount of the labeled antigen, fluorescence polarization values increasing in parallel with each addition of antibody. Antigen content was determined with a known amount of antibody, which reacted at first with an unknown amount of antigen in samples, followed by addition of a definite amount of the labeled antigen (competition). I used the method to determine a pharmacokinetic parameter, the apparent volume of distribution for neocarzinostatin in rabbits, using drug-injected rabbit sera. I evaluated precision, accuracy, and reproducibility, using various samples or possible interfering substances such as bilirubin and hemoglobin, and also compared results for antigen with those by single radial immunodiffusion assay. The present assay is fast (less than 2 min), sensitive (less than 10 nmol/liter can be detected), and simple (there is no separation step before readout of the results).

Animals↗
PubMed1978

Differentiation between patients with malignant diseases and non-malignant diseases or healthy donors by changes of fluorescence polarization in the cytoplams of circulating lymphocytes.

Changes in the degree of fluorescence polarization in the fluorescent cytoplasm of lymphocytes from patients with malignant diseases, patients with nonmalignant diseases, and normal donors were investigated by the fluorescence polarization technique. In order to confirm Cercek's cancer test, two kinds of parameters, the degree of fluorescence polarization of lymphocytes stimulated with phytohemagglutinin (PHA) and cancer basic protein, were obtained for each person. Patients with malignant diseases were easily differentiated from the others without exception by an index, the response ratio of the degree of fluorescence polarization. Usefulness of this technique was confirmed for the detection of cancer-bearing patients.

Adolescent↗
PubMed1979

Fluorescence polarization immunoassay for cortisol.

A fluorescence polarization immunoassay for serum cortisol was established using cortisol 21-amine which was readily coupled with fluorescein isothiocyanate. The proposed method is sufficiently sensitive, reliable, specific and simple for routine determination of serum cortisol. The assay is rapid, without separation of antibody-bound and free ligands. The minimal amount of cortisol detected was 1.5 ng/tube and the measurable range was from 1.5 to 100 micrograms/dl. There was a good correlation between values obtained from radioimmunoassay and the proposed method.

Cross Reactions↗
PubMed1977

The influence of temperature and urea on intrinsic fluorescence of Streptomyces subtilisin inhibitor. A study by fluorescence polarization and quenching.

Intrinsic fluorescence of new microbial protease inhibitor, Streptomyces subtilisin inhibitor was studied by observing fluorescence polarization degree and lifetime in the temperature range 25-81 degrees C. Striking thermal changes in these fluorescence properties of tryptophan residues were observed. The apparent molecular volumes for tryptophan and tyrosine residues in the native form were determined to be 89 and 75 A3, respectively. The fluorescence quenching by Br- or Cs+ was investigated to obtain a microenvironmental information around tryptophan residues both in the native and denatured form. Cs+ quenches the fluorescence slightly stronger than Br-, implying that there is not any distinctive electrostatic interaction between tryptophan residues and their neighborhood.

Bacterial Proteins↗
PubMed1976

Anisotropy of photosynthetic membranes and the degree of fluorescence polarization.

The degree of fluorescence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400-680 nm) and emission wavelengths (675-750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, PIN and PAN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization PIN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Qy (approximately 660-680 nm) absorption band of chlorophyll in vivo. With 670-680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts PIN is shown to be +5+/-1%. The excitation wavelength dependence studies of PIN indicate that the carotenoid and chlorophyll Qy transition moments tends to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).

Cell Membrane↗
PubMed1977

Fluorescence polarization studies of rat intestinal microvillus membranes.

Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin.

Animals↗
PubMed1976

Changes in rotational motion of a cell-bound fluorophore caused by colicin E1: a study by fluorescence polarization and differential polarized phase fluorometry.

The stationary fluorescence polarization and the differential phase delay of the polarized components of the fluorescence of 1-phenylnaphthylamine in Escherichia coli suspensions were measured before and after addition of colicin E1. Both sets of measurements register an increase in the rotational relaxation time of the fluorescent probe when colicin is present. These increases are absent in an E. coli mutant tolerant to colicin E1. The physical interpretation of the changes demands separate estimation of the fraction f2 of the emitting fluorophores that change their properties upon colicin addition and of the rotational relaxation time p2 of this fraction, following the colicin-induced changes. By themselves, the steady state polarizaiton observations permit only the conclusion that f2 must be in the range of 1-0.06 and the change in p2/pi between 1.5 and a value larger than 10. Combination of the data of stationary polarization with those of differential phase fluorometry results in an important reduction in the uncertainty:f2 must be in the range 1-0.33 and the change in p2/pi in the range 1.5-2.5.

Binding Sites↗
PubMed1979

Amniotic fluid microviscosity determined by fluorescence polarization: methodology and relation to gestational age.

The fluorescence polarization of 116 amniotic fluid specimens obtained from 22 isoimmunized pregnant women was determined. The degree of fluorescence polarization of amniotic fluid provides an index of microvisocity in lipid aggregates that is dependent on the lecithin-to-sphingomyelin ratio and the degree of saturation of fatty acid side chains. We confirmed the reproducibility of the measurement of amniotic fluid microviscosity (coefficient of variation, 2.0%). The measurements are not effected by bilirubin concentration of amniotic fluid dilution. The pattern of change of amniotic fluid microviscosity during gestation parallels the expected development of the surfactant system. Amniotic fluid microviscosity is high during early gestation and abruptly and sequentially decreases between the 28th and 36th week of gestation. Since the measurements are an accurate reflection of the biochemical properties of amniotic fluid lipids and parallel the development of the surfactant system, we conclude that amniotic fluid microviscosity may well serve as an indicator of the process of fetal lung maturation.

Amniotic Fluid↗
PubMed1977

A theory of fluorescence polarization decay in membranes.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.

Liposomes↗
PubMed1978

Determination of fetal lung maturity by fluorescence polarization of amniotic fluid.

The degree of fluorescence polarization (P value) of 1,6-diphenyl-1,3,5-hexatriene has been shown to be an excellent indicator of the lecithin/sphingomyelin (L/S) ratio in laboratory prepared liposomal dispersions. In order to test the validity of this technique in amniotic fluid, the L/S ratio and P value of 161 amniotic fluid specimens were determined. In 127 the P value was less than or equal to 0.336 and the L/S ratio was greater than or equal to 2. In 26 the P value was greater than 0.336 and the L/S ratio was less than 2 (P less than 0.05). The amniotic fluid P value is affected by blood or meconium but not by surgical lubricants or bilirubin. We concluded that the degree of fluorescence polarization of amniotic fluid indicates fetal lung maturity as reliably as the L/S ratio. However, this method has important technical and practical advantages that make it more applicable for a clinical laboratory.

Amniotic Fluid↗
PubMed1976

Physical properties of muscle cell membranes during fusion. A fluorescence polarization study with the ionophore A23187.

1. The fluorescence polarization of A23187 is used to detect physical changes in myoblast membranes produced by Ca2+ concentrations which are able to trigger fusion. Temperature scans reveal a Ca2+-dependent fluidity increase in the microenvironment of the ionophore above 37 degree C. 2. Time-dependent polarization measurements show two different effects. The first one consists of a fast polarization increase which reaches its maximum after 5-10 min. This change could be explained by a Ca2+-induced phase separation of acidic and neutral phospholipids in the membrane. The second effect is a slow polarization decrease over 2-3 h subsequent to the fast increase. 3. The information derived from the fluorescence polarization is in accordance with the characteristics of the fusion of myoblast cells regarding the dependence on Ca2+ concentration, the inhibition by Mg2+ and lysolecithin as well as a typical temperature of 35-37 degree C where fusion rate changes abruptly.

Animals↗
PubMed1976

Fluorescence polarization measurement of the hormone-binding site interaction.

Fluorescence polarization methodology has been applied to the binding of fluorescent-labeled prolactin, growth hormone and estradiol to subcellular fractions prepared from rabbit mammary and uterine tissue. Equilibrium measurements treated by Scatchard plots have shown that there are high affinity sites (K approximately 10(9) 1 mol-1), as well as lower affinity sites (K approximately 10(8) 1 mol-1) for both hormones. The binding of the fluorescent labeled hormone to microsomal or cytosol fractions has been shown to be inhibited by the prior addition of native, unlabeled hormone. Kinetic results on the interaction of prolactin with the microsomal fraction are consistent with a bimolecular reaction involving significant structural rearrangements during the reaction (not diffusion controlled). The forward rate constant calculated from data on initial rates was found to be 1.7 X 10(5) 1 mol-1 sec-1. Stopped flow kinetic measurements on the reaction between fluorescent-labeled estradiol and cytosol binding sites show that at low temperatures, the reaction goes in two distinct steps separable in time. The second step may be the reaction found by others (utilizing sedimentation velocity methods) which precedes translocation of the hormone-binding site complex to the nucleus. Fluorescence polarization makes it possible to observe both the formation and dissociation of hormone-binding site complexes over a time scale down to a fraction of a second and at concentrations down to the nanogram per nl range.

Animals↗
PubMed1975

Structure and function of D-amino acid oxidase. IX. Changes in the fluorescence polarization of FAD upon complex formation.

1. The fluorescence polarization, P, of FAD increased on complex formation with the apoenzyme of D-amino acid oxidase [D-amino acid: O2 ocidoreductase (deaminating), EC 1.4.3.3]. The time course of the increase was monophasic. The values of P were extimated to be 0.04, 0.4, and 0.4 for FAD, the enzyme and the enzyme-benzoate complex, respectively. 2. The value of P of the enzyme is dependent on its concentration, indicating that the degrees of dissociation of FAD in the monomer and dimer are different. The dissociation constant was calculated to be 7 times 10-minus 7 M for the monomeric form of the enzyme. This value is far larger than the value for the dimeric form of the enzyme, 1 times 10-minus 8 M, calculated from equilibrium dialysis data. 3. Changes in fluorescence polarization of the enzyme due to changes in solution pH or temperature can be explained in terms of the monomer-dimer equilibrium.

Animals↗
PubMed1979

Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization.

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.

Carbocyanines↗