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At least 19 recordsLinked to original sources
PubMed1991

Fluorescence polarization and low-temperature absorption spectroscopy of a subunit form of light-harvesting complex I from purple photosynthetic bacteria.

Measurements of polarized fluorescence and CD were made on light-harvesting complex 1 and a subunit form of this complex from Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodobacter capsulatus. The subunit form of LH1, characterized by a near-infrared absorbance band at approximately 820 nm, was obtained by titration of carotenoid-depleted LH1 complexes with the detergent n-octyl beta-D-glucopyranoside as reported by Miller et al. (1987) [Miller J. F., Hinchigeri, S. B., Parkes-Loach, P. S., Callahan, P. M., Sprinkle, J. R., & Loach, P. A. (1987) Biochemistry 26, 5055-5062]. Fluorescence polarization and CD measurements at 77 K suggest that this subunit form must consist of an interacting bacteriochlorophyll a dimer in all three bacterial species. A small, local decrease in the polarization of the fluorescence is observed upon excitation at the blue side of the absorption band of the B820 subunit. This decrease is ascribed to the presence of a high-energy exciton component, perpendicular to the main low-energy exciton component. From the extent of the depolarization, we estimate the oscillator strength of the high-energy component to be at most 3% of the main absorption band. The optical properties of B820 are best explained by a Bchl a dimer that has a parallel or antiparallel configuration with an angle between the Qy transition dipoles not larger than 33 degrees. The importance of this structure is emphasized by the results showing that core antennas from three different purple bacteria have a similar structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriochlorophylls↗
PubMed1992

[A comparative analysis of the immunochemical determination of gentamycin by fluorescence polarization and quenching].

A polarization fluorescence immunoassay (PFIA) for gentamicin with using a set of reagents made in this country was developed. One ml of fluorescein-labeled gentamicin (50 nM) and 100 microliters of antiserum are added to 50 microliters of the sample and the fluorescence polarization is measured. The time of the assay is 10 to 15 minutes, the range of the measurable concentrations is 0 to 800 ng/ml, the sensitivity of the method is 5 ng/ml and the accuracy is 5.8-10.3 per cent. A fluorescence quenching immunoassay (FQIA) for gentamicin was also developed. Determination of gentamicin by the FQIA does not require the use of a specific polarization fluorimeter. Its linear calibrating dependence is more convenient. However, its accuracy and sensitivity are 3 times lower than those of the PFIA.

Drug Monitoring↗
PubMed1987

[Intravital study of the hydrophobic interactions in protein fibrils by a polarization-fluorescence method. I. Collagen fibrils].

It has been shown earlier that anisotropy of extrinsic fluorescence (AEF) of ordered structures in living cell may serve as a measure of the free structure energy. The experimental study of AEF changes of myelin at varying temperatures (0-30 degrees C) revealed cold denaturation. This phenomenon occurs only in cases when the structure in question is stabilized through hydrophobic interactions. The purpose of this work was to show that not only membranes but also some native ordered protein structures might be analysed by fluorescence polarization to detect hydrophobic interactions. The fluorescence anisotropy of primulin adsorbed by collagen fibrils from rat tail has been investigated. The fluorophore orientation decreases with temperature, i.e. here the phenomenon of cold denaturation is observed. When the medium humidity falls down to 80%, no cold denaturation occurs. AEF of collagen fibrils depends to a considerable extent on organic substances (ethanol, ethylene glycol) added in small concentrations to the incubation medium. It is concluded that the dependence of the whole collagen molecular structure in fibrils on the content and structure of the solvent shell is of critical nature.

Animals↗
PubMed1986

Determination of fetal lung maturity in rats by fluorescence polarization.

The fluorescence polarization technique was utilized to determine fetal lung maturity in rats. The study confirmed that changes in fluorescence polarization values with gestational age follow the pattern already seen in human beings. A sharp drop was observed on the afternoon of the 20th day of gestation which corresponds in the rat to the beginning of surfactant synthesis. This was confirmed by determination of phosphatidylglycerol. In order to verify the effectiveness of this animal model, a group of pregnant rats was treated with 100 micrograms/kg betametasone twice 48 and 24 hr before the 20th or 21st days of gestation. As expected, this significantly reduced fluorescence polarization on day 20 (4-6 p.m.) and day 21 of gestation, indicating increased surfactant synthesis in betametasone-treated rats. Another group of female rats was pretreated before mating with streptozotocin (40 mg/kd), inducing diabetes. Fluorescence polarization values in the amniotic fluid of the diabetic rats at the above intervals were significantly higher than controls, indicating reduced lung maturation.

Amniotic Fluid↗
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↗
PubMed1986

Change of intracellular fluidity during keratinocyte differentiation measured by fluorescence polarization.

The fluorescence polarization method was applied to measure the intracellular fluidity of fractionated guinea pig keratinocytes. Guinea pig epidermal cell suspension was obtained by treatment with EDTA and trypsin, and was separated into high, intermediate, and low density fractions using Percoll density gradient centrifugation. Morphological observation and cytofluorometric analysis of DNA content in the fractionated epidermal cells showed that the high, intermediate, and low density fractions were basal, spinous, and granular cell-rich fractions, respectively. Intracellular fluorescence polarization of each fraction was determined by a polarization spectrofluorometer (Hitachi MPF-4, prototype) with fluorescein diacetate. The P-values were calculated for high, intermediate, and low density fractions as 0.192 +/- 0.021, 0.172 +/- 0.019, and 0.147 +/- 0.012, respectively. Since low P-values indicate a high degree of fluidity, the results indicate that intracellular fluidity of keratinocytes is lower in basal cells and higher in granular cells. Dye-binding experiments showed that fluorescein-binding proteins were not detected in the soluble fraction of the epidermal cells. The present findings suggest that intracellular fluidity of the guinea pig keratinocyte increases during the process of its differentiation.

Animals↗
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↗
PubMed1988

Measurement of angiotensinogen in human serum by fluorescence polarization immunoassay.

Fluorescence polarization immunoassay (F.P.I.A.) has rarely been used to measure components of the renin system. Using an Abbott TDX polarimeter and fluorescein - labelled angiotensin I as a tracer we measured angiotensin I by F.P.I.A. Combining this procedure with a renin incubation step enabled measurement of angiotensinogen in human serum. Using sera from male patients and from pregnant females, a good correlation between radioimmunoassay (R.I.A.) and F.P.I.A. was found. Two procedures were developed; one involving taking samples from the renin incubation mixture, the other involving measurement of generated angiotensin I at intervals without interrupting the renin incubation procedure. F.P.I.A. is less expensive and somewhat simpler than R.I.A. but, with the instrument used, it was less sensitive. An unexpected observation was that human renin increases polarization in solutions containing fluorescein labelled angiotensin I, indicating possible binding of renin to angiotensin I.

Angiotensin I↗
PubMed1988

Measurement of protein rotational motion using frequency domain polarized fluorescence depletion.

Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are examined anaerobically in a fluorescence microscope. In time domain PFD experiments a several-microsecond pulse of linearly polarized light produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. Monitoring the decay of ground state depletion with a probe beam alternatively polarized, parallel, and perpendicular to the depletion pulse permits the triplet lifetime and rotational correlation time to be resolved and evaluated. We have now explored fluorescence depletion methods in the frequency domain to see whether such measurements could provide simpler and more efficient routine measurements of protein rotational relaxation than previous time domain PFD methods. An acousto-optic modulator (AOM) modulates the intensity of a 514.5 nm argon ion laser beam and a Pockels cell (PC) rotates its plane of polarization. These devices are driven by sinusoidal or square waves in fixed frequency relation, and rigidly phase locked, one to another. The fluorescence emitted from a sample then contains various overtones and combinations of the AOM and PC frequencies. The magnitude and phase of individual fluorescence signal frequencies are measured by a lock-in amplifier using a reference also phase-locked to both the AOM and PC. Specific frequencies permit evaluation of the rotational correlation time of the macromolecule and of the fluorophore triplet state lifetime, respectively. Measurement of bovine serum albumin rotation in glycerol solutions by this method is described.

Mathematics↗
PubMed1985

[Structural changes in the myelin sheath membranes exposed to anisotonic media. A fluorescence polarization study].

Extrinsic fluorescence polarization (P parallel and P perpendicular) of intact myelinated nerve fibres in anisotonic media has been investigated. Fluorescent probes of Acridine orange and Perylen were used for the experiment. Changes in fluorescent polarization of nerve fibres, associated with osmotic movement of water, arise immediately after the incubation of fibres in hypo- or hypertonic solutions. During exosmotic and endosmotic movement of water P parallel and P perpendicular and in quite a different manner. This fact demonstrates an asymmetry of structural membrane alterations. The results obtained are explained by asymmetry of swelling and shrinking of carbohydrate gel between two adjacent extracellular membrane surfaces of the myelin sheath.

Animals↗
PubMed1990

POLAR: a menu driven computerized analysis of fluorescence polarization data.

While fluorescence polarization is a powerful tool for the measurement of ligand-receptor (e.g. antigen-antibody) interactions, its application in routine clinical chemistry and immunochemistry has been limited because collection of data was error prone, and because analysis of data previously required tedious and imprecise manual graphical methods. We present the series of linear and curvilinear regressions required to calculate parameters (p', Q', pf, Qf, Pb, Qb, Fb max, a and Ko) and their corresponding standard errors. The values of a and Ko are comparable to those obtained by the method of finding the minimal the sum of squares. A menu operated computer program that uses these regressions, simplifies the collection, analysis and presentation of fluorescence polarization data. A simple spread sheet system permits the insertion, deletion or modification of data. The results are presented in graphical and tabular form, either on a monitor, a printer or an X-Y plotter. The system has its own graphical routines.

Algorithms↗
PubMed1990

Adaptation of fluorescence polarization immunoassay to the assay of macromolecules.

This paper describes an original methodology for determining macromolecular antigen levels by polarization of fluorescence. it involves the use of fluorescent derivatives of Fab fragments of a monoclonal antibody (Mr 50,000), whose fluorescence polarization rises significantly when it combines with a macromolecular antigen. An experimental system (Fab anti-aldosterone and aldosterone--bovine serum albumin (BSA)) is studied to test this methodology, which was then used to develop an immunoassay for human immunoglobulin M (IgM), using anti-mu chain Fabs. In the two assays, the binding stoichiometry of Fab/antigen was 10/1 and 8/1 for aldosterone--BSA and IgM, respectively. The lower limit of detection of the IgM assay was 0.8 microgram/ml and thus it was applicable to clinical detection of IgM concentrations.

Aldosterone↗
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↗
PubMed1990

Slow rotational mobilities of antibodies and lipids associated with substrate-supported phospholipid monolayers as measured by polarized fluorescence photobleaching recovery.

Polarized fluorescence photobleaching recovery has been used to monitor slow rotational motions of a fluorescently-labeled anti-dinitrophenyl mouse IgGl monoclonal antibody (ANO2) specifically bound to substrate-supported monolayers composed of a mixture of distearoylphosphatidylcholine (DSPC) and dinitrophenyldioleoylphosphatidylethanolamine (DNP-DOPE). ANO2 antibodies were labeled with a new bifunctional carbocyanine fluorophore that has two amino-reactive groups; steady-state fluorescence anisotropy data confirmed the expected result that the ANO2-conjugated bifunctional probe had less independent flexibility than ANO2-conjugated unifunctional fluorescence labels. Rotational mobilities were also measured for the fluorescent lipid 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine (dil) in DSPC and in mixed DSPC/DNP-DOPE monolayers in the presence and absence of unlabeled ANO2 antibodies. The apparent rotational correlation time and fractional mobility of ANO2 on supported monolayers were approximately 70 and approximately 0.3 s, respectively. These measured parameters of rotational mobility did not depend on the ANO2 surface density or on kinetic factors, but addition of unlabeled polyclonal anti-(mouse IgG) antibodies significantly decreased the apparent mobile fraction. The measured fluorescence recovery curves for dil were consistent with two fluorophore populations with rotational correlation times of approximately 4 and approximately 100 s and a population of immobile fluorescent lipid. No difference in fluorescence recovery and decay curves was measured for dil in DSPC monolayers, DSPC/DNP-DOPE monolayers, and DSPC/DNP-DOPE monolayers treated with unlabeled ANO2 antibodies.

Animals↗
PubMed1988

[Intravital research on hydrophobic interactions in protein fibrils using a fluorescence polarization method].

Fluorescent probe of acridine orange (AO) is known to be bound exclusively by materials of thick protofibril muscle fibres. The dependence of fluorescence anisotropy of AO in frog muscle fibres on the temperature has been studied. It has been found that the fluorophore orientation decreases with temperature, i.e. here the adsorbent cold denaturation takes place. This phenomenon was demonstrated for living, skinned and glycerinated muscle fibres. It shows that hydrophobic interactions contribute much to stabilization of the myosin structure. After contraction of glycerinated muscle fibres at the action of ATP, the AO fluorescence anisotropy grows independently of the temperature which is indicative of structural rearrangements in materials of thick protofibrils upon contraction.

Acridine Orange↗
PubMed1989

Influence of auranofin on lymphocyte membrane fluidity in rheumatoid arthritis. A fluorescence polarization study.

Using the fluorescence polarization of 1.6-diphenyl-1, 3, 5-hexatriene, the membrane fluidity of lymphocytes obtained from rheumatoid arthritis patients was measured during 12 months of auranofin treatment. Significant differences in fluorescence polarization values were observed following auranofin treatment. The correlations shown between fluorescence polarization values and the indices of rheumatoid arthritis activity, such as erythrocyte sedimentation rate, alpha 2-globulins, plasma fibrinogen levels and platelet count suggest that changes in lymphocyte membrane fluidity could be used to monitor the effects of auranofin therapy.

Adult↗
PubMed1988

The influence of abnormal pregnancies on fluorescence polarization of amniotic fluid lipids.

The fluorescence polarization of amniotic fluid, a measure of fetal lung maturity, was determined in 518 amniotic fluid samples obtained by amniocentesis. The subjects were divided into seven clinical groups: premature contractions, premature rupture of the membranes, pregnancy-induced hypertension, diabetes, intrauterine growth retardation, vaginal bleeding, and "other" for gestational-age groups of 30 weeks or less, 31-36 weeks, and 37 or more weeks. The proportion of mature values (fluorescence polarization 0.285 or lower) increased progressively from 12.5% at 27-28 weeks to 100% at 39-40 weeks. In the 31-36-week gestation group, the proportion of mature values in subjects with premature rupture of the membranes (84.6%) was significantly higher than in those with premature contractions (60%), severe pregnancy-induced hypertension (50%), mild pregnancy-induced hypertension (55.2%), diabetes class A (50%), insulin-dependent diabetes (60%), and other (63.5%). The mean +/- SD fluorescence polarization value was significantly lower in premature rupture of the membranes (0.256 +/- 0.030) than in premature contractions (0.274 +/- 0.032), mild and severe pregnancy-induced hypertension (0.280 +/- 0.027 and 0.280 +/- 0.035, respectively), and class A and insulin-dependent diabetes (0.285 +/- 0.018 and 0.277 +/- 0.030, respectively). The severity of pregnancy-induced hypertension and diabetes did not appear to influence either the fluorescence polarization value or the proportion of mature results. With the exception of a marked influence of premature rupture of the membranes, abnormal pregnancy conditions did not appear to have a significant effect on fluorescence polarization of amniotic fluid.

Amniotic Fluid↗
PubMed1975

Degree of dissociation of apohemoglobin studied by nano-second fluorescence-polarization technique.

A fluorescent dye 1-anilino-8-naphthalene sulfonate was complexed with human apohemoglobin and sperm whale apomyoglobin. Nanosecond fluorescence-polarization kinetics were measured for each of these complexes in KC1 solutions to obtain their fluorescence lifetimes and rotational correlation times. The rotational correlation time of apohemoglobin-dye complex was found to be 21 ns, which was about twice that of apomyoglobin-dye complex, 11 ns. These values were constant over an ionic strength range from 0 to 1.7. Circular dichroism spectra (215-300 nm) and fluorescence lifetimes of the complexes were also found to be independent of the ionic strength, indicating that no gross conformational change occurs with the change in the salt concentration, These results suggest that apohemoglobin remains dimeric over the ionic-strength range examined.

Anilino Naphthalenesulfonates↗