Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin.

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.

Kappa-squared: from nuisance to new sense.

The orientation factor, which is commonly called kappa-squared, is often considered to be a nuisance because it represents a significant uncertainty in the distance obtained with the FRET technique. It is shown that this uncertainty is rather small in almost all cases of practical interest if one takes the width of a 67% confidence interval (CI) for the distance distribution as a measure of uncertainty. Kappa-squared has the potential to open up new information on orientations and rotations from time-resolved studies of donor and acceptor anisotropies. One can make sense of such data by designing matrix models for the transitions between states describing various orientations and positions of donors and acceptors in the system.

Comparison of photovoltaic behaviors for horseradish peroxidase and its mimicry by surface photovoltage spectroscopy.

Surface photovoltage spectroscopy (SPS) was chosen to study the photovoltaic behavior of horseradish peroxidase (HRP), hemin and immobilized hemin (poly(NIPAAm/MBA/hemin)). Different photovoltaic behaviors were observed in these three systems. In air, similar SPS curves were found for HRP and poly(NIPAAm/MBA/hemin) with different response intensities. However, poly(NIPAAm/MBA/hemin) showed a wider changing range upon increasing the positive and negative bias to 1.0 V. The SPS of hemin showed a total different behavior when an external positive potential was applied. In vacuum, clearly different photovoltaic behaviors were found. Moreover, the response value decreased when HRP was exposed to O2, the SPS intensity was different from that in air, and could be altered by changing the external biases. On the other hand, the SPS could not be changed before and after poly(NIPAAm/MBA/hemin) was exposed to O2. These differences may result from different chemical microenvironments for hemin in HRP versus that in poly(NIPAAm/MBA/hemin). It could be concluded that H2O and O2 were important factors affecting the photovoltage response in HRP, but only H2O played this important role in poly(NIPAAm/MBA/hemin).

Enhancement of the emission intensity of fluorophore-labeled avidin by biotin and biotin derivatives. Evaluation of different fluorophores for improved sensitivity.

Fluorescein, Texas Red, Cascade Blue, 7-amino-methylcoumarin-3-acetic acid, and Lucifer Yellow were evaluated as fluorescent labels for homogeneous fluorophore-linked binding assays. Conjugates of avidin with these fluorophores exhibited an enhancement in fluorescence emission in the presence of biotin or biotin derivatives. This property was used in the development of assays for biotin. The biotin-induced fluorescence enhancement of each labeled avidin were compared. Fluorescein led to the most sensitive calibration (dose-response) curve for biotin with a detection limit of 8 x 10(-10)M.

Designing matrix models for fluorescence energy transfer between moving donors and acceptors.

A recipe is given for designing theoretical models for donor-acceptor systems in which fluorescence energy transfer and motion takes place simultaneously. This recipe is based on the idea that a system exhibiting both motion and fluorescence energy transfer can be modeled by specifying a number of "states" and the rates of transitions between them. A state in this context is a set of specific coordinates and conditions that describe the system at a certain moment in time. As time goes on, the coordinates and conditions for the system change, and this evolution can be described as a series of transitions from one state to the next. The recipe is applied to a number of example systems in which the donors and/or acceptors undergo either rotational or translational motion. In each example, fluorescence intensities and anisotropies for the donor and acceptor are calculated from solutions of eigensystems. The proposed method allows for analyzing time-resolved fluorescence energy transfer data without restrictive assumptions for motional averaging regimes and the orientation factor. It is shown that the fluorescence quantities depend on the size of the motional step (i.e., on the number of states), only if fluorescence energy transfer occurs. This finding indicates that fluorescence energy transfer studies may reveal whether the dynamics of a system (e.g., a protein) is better described in terms of transitions between a relatively small number of discrete states (jumping) or a large number of dense states (diffusion).

[The clinical significance of anti-gliadin antibodies determination in blood of children with suspected celiac diseases]. / De klinische waarde van de bepaling van anti-gliadine antilichamen in het serum van kinderen met verdenking van coeliakie.

Screening tests for coeliac disease may be useful to select the patients who should undergo a small intestine biopsy. It has been reported that the determination of serum levels of anti-gliadin antibodies is a good screening method for small intestine damage in coeliac disease. We have retrospectively assessed the value of the anti-gliadin antibody test in detecting villous atrophy of the small bowel. Antigliadin antibodies were measured with an ELISA technique in sera of 44 children seen at the Department of Paediatrics, Leiden University Hospital, who underwent 53 small intestine biopsies because of suspected coeliac disease. The relation between the histopathological findings of the small intestine and the results of the anti-gliadin antibody quantifications in serum were studied. In 9 of the 10 children with villous atrophy high Ig total and IgG-titers were found. Five children with villous atrophy, 2 of them with a selective IgA-deficiency, had normal IgA-AGA titers in serum. 17 Children with normal biopsies had high Ig total and IgG-AGA titers, but no child with normal small intestine biopsy had high IgA-AGA titers in serum. We conclude that the determination of anti-gliadin antibodies as screening for mucosal damage must be based on the measurement of antibodies within the IgG as well as the IgA class.

Theory of two-photon induced fluorescence anisotropy decay in membranes.

We report the first theoretical description for the time-dependent fluorescence anisotropy decay resulted from two-photon excitation (r([2])(t)) for fluorophores in macroscopically isotropic and oriented membranes. In case of two-photon excitation, the initial value of the fluorescence anisotropy r([2])(0) immediately after excitation by a flash of polarized light is a function of the components of the two-photon absorption transition tensor [unk]S and the projections of the emission transition moment to the principal axes of [unk]S. The components of [unk]S depend on the symmetries of all molecular states relevant to the two-photon absorption process. The maximal value of r([2])(0) is proven to be as large as 0.61 in contrast to 0.4 for the conventional one-photon induced fluorescence anisotropy r([1])(0). It is shown that only for some special cases the ratio of the two-photon r([2])(t) over the conventional one-photon r([1])(t) will be a constant at all times for fluorophores in macroscopically isotropic membrane systems. In oriented membrane systems, an additional order parameter can be determined by the use of angle-resolved fluorescence depolarization measurements resulted from two-photon excitation. The advantages of measuring time-resolved fluorescence anisotropy decays or angle-resolved fluorescence depolarization ratios by two-photon excitation for the study of orientational dynamics in isotropic or oriented membranes are discussed from the theoretical point of view.

Quantitation of lateral stress in lipid layer containing nonbilayer phase preferring lipids by frequency-domain fluorescence spectroscopy.

Frequency-resolved fluorescence measurements have been performed to quantitate the lateral stress of the lipid layer containing nonbilayer phase preferring dioleoylphosphatidylethanolamine (DOPE). On the basis of a new rotational diffusion model, the wobbling diffusion constant (Dw), the curvature-related hopping diffusion constant (DH), and the two local orientational order parameters ([P2] and [P4]) of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC) in fully hydrated DOPE and DOPE/dioleoylphosphatidylcholine (DOPC) mixtures were calculated from the frequency-domain anisotropy data. The values of [P2], [P4], and DH for DOPE were found to increase significantly at approximately 12 degrees C, the known lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition temperature of DOPE. Similar features as well as a decline of Dw were detected in the DOPE/DOPC mixtures as the DOPE content was increased from 85% to 90% at 23 degrees C, corresponding to the known lyotropic phase transition of the DOPE/DOPC. In contrast, for DOPC (0-40 degrees C) and DOPE/DOPC (0-100% DOPE at 3 degrees C), which remained in the L alpha phase, these changes were not detected. The most probable local orientation of DPH-PC in the DOPE/DOPC mixtures shifted progressively toward the normal of the lipid/water interface as the content of DOPE increased. We concluded that the curvature-related lateral stress in the lipid layer increases with the content of the nonbilayer phase preferring lipids.

Intramolecular excimer formation of pyrene-labeled lipids in lamellar and inverted hexagonal phases of lipid mixtures containing unsaturated phosphatidylethanolamine.

The rates of intramolecular excimer formation of di(1'-pyrenemyristoyl)phosphatidylcholine (dipyPC) in dioleoylphosphatidyl-ethanolamine (DOPE), egg PE/diolein (DG) and dilinoleoyl-PE (DLPE)/1-palmitoyl-2-oleoyl-PC (POPC) were studied at different temperatures and lipid compositions. Both the excimer-to-monomer intensity ratio and the excimer association rate constant were employed to quantify the rate of excimer formation. The latter was calculated from the measured monomer fluorescence lifetime of dipyPC. We observed that the rate of excimer formation was sensitive to either the temperature-induced or lipid composition-induced lamellar-to-inverted hexagonal phase transition of the above lipid systems. As the lipids entered the inverted hexagonal phase, the rate of excimer formation increased at the temperature-induced phase transition for DOPE, but decreased at the composition-induced phase transition for both TPE/DG and DLPE/POPC systems by increasing the DG% and decreasing the PC%, respectively. We conclude that the rate of intramolecular excimer formation of dipyPC in the non-lamellar phase is influenced both by the intra-lipid free volume of the hydrocarbon region and the intra-rotational dynamics of the two lipid acyl chains.

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. I. Theory.

It is shown that fluorescence anisotropy from lipidlike probes in the hexagonal HII phase gives information of (a) orientational order parameters, (b) the wobbling diffusion constant, and (c) the hopping diffusion constant of the probe, DH, equals DL/R2, the lateral diffusion constant over the square of the radius of the hexagonal tubes. Here we consider only lipidlike probes having the absorption transition movement and/or the emission transition moment along the long axis of the molecule. Three models are introduced for analysis of time-resolved data: the "WOBHOP," the "reduced WOBHOP," and the "P2P4HOP" model. The fluorescence anisotropy in response to a very short excitation pulse in each of the three models is a constant plus a number of exponentials. The WOBHOP and reduced WOBHOP models have 3 and 2 exponentials, respectively, and both contain four fitting parameters: r0 (the fundamental anisotropy), (P2) (the second rank orientational order parameter), DW (the wobbling diffusion constant), and DH (the hopping diffusion constant). The P2P4HOP model has eight exponentials and five fitting parameters: the four parameters listed above and (P4) (the fourth rank orientational order parameter). Analysis of fluorescence anisotropy data in the hexagonal HII phase using one of these models allows for obtaining the hopping diffusion constant, and, if the lateral diffusion constant is known, the radius of the hexagonal tubes. Substitution of DH = 0 in each of the three models yields an expression for the fluorescence anisotropy that is used in the literature for lamellar (L alpha or L beta) phases. The fluorescence anisotropy in coexisting L alpha/HII phases is discussed.

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. II. An experimental study.

The polymorphic phase behavior of unsaturated phosphatidylethanolamine (PE)/diacylglycerol (DG) binary lipid mixtures was investigated by the use of time-resolved fluorescence anisotropy. Using a fluorescent lipid, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenylethyl] carbonyl]3-sn-phosphatidyl-choline (DPH-PC), the orientational order and rotational dynamics of the above lipid mixtures in the liquid crystalline lamellar (L alpha) and inverted hexagonal (HII) phases were studied. By employing a one-exponential model (Cheng, K.H. 1989: Biophys. J. 55:1025-1031) to fit the anisotropy decay data, abrupt decreases in the normalized initial anisotropy decay slope and the residual anisotropy of DPH-PC were observed at approximately 6-8% DG, signifying a L alpha/HII phase transition. Using our new theoretical WOBHOP and P2P4HOP models as described in a preceding paper (Van Der Meer, B.W., K.H. Cheng, and S.Y. Chen. 1990. Biophys. J. 58:000-000), two or more rotational correlation times were required to describe the anisotropy decay behavior of DPH-PC in the HII phase. These rotation correlation times were further related to the second and fourth rank order parameters, and the wobbling and hopping diffusion constants of the fluorescent probe in the highly curved lipid cylindrical tubes of the HII phase. The hopping diffusion constant (DH) equals the lateral diffusion constant (DL) divided by R2 (R = radius of the lipid cylindrical tubes). The value of DL was estimated by measuring the excimer formation rate of 1-palmitoyl-2-[10-(1-pyrenl)decanoyl] phosphatidyl choline (py-PC) in the same PE/DG mixtures. Upon comparing the values of DH and DL, the value of R was determined to be approximately 10-15 A, and agreed with that derived from x-ray diffraction (Tate, M.W., and S.M. Gruner, 1989, Biochemistry. 28:4245-4253; Rand, R.P., N.L. Fuller, S.M. Gruner, and V.A. Parsegian. 1990. Biochemistry. 29:76-87).

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.

Role of BBM lipid composition and fluidity in impaired renal Pi transport in aged rat.

Renal tubular phosphate (Pi) transport is impaired in the aged rat. The decrement in Pi transport is manifested as a decrease in the Vmax of the luminal brush-border membrane (BBM) sodium-dependent Pi transport. In the present study we determined the potential role of alterations in cortical BBM lipid composition and fluidity in the age-related phosphaturia observed in rats. In the aged rat there are significant increases in the BBM cholesterol (Chol, 504 vs. 422 nmol/mg protein in adult, P less than 0.01) and sphingomyelin (Sph, 41.8 vs. 37.5 mol% in adult, P less than 0.01) and a decrease in the BBM fluidity [increase in fluorescence anisotropy of diphenylhexatriene (DPH), rDPH, 0.221 vs. 0.215 in adult, P less than 0.01]. The BBM lipid compositional and fluidity alterations may also play an important role in the impaired renal adaptation to a low-Pi diet in the aged rat. In the adult rat the renal adaptation to a low-Pi diet is associated with a decrease in BBM Chol (370 to 307 nmol/mg protein, P less than 0.01) and an increase in fluidity (decrease in rDPH 0.207 to 0.201, P less than 0.05). In the aged rat the renal adaptation to a low-Pi diet is incomplete and is associated with impaired ability to lower BBM Chol (441 to 429 nmol/mg protein, P = NS) and to increase fluidity (rDPH 0.211 to 0.211, P = NS). The results of this study therefore suggest that in the aged rat the increase in BBM Chol and Sph content, and the decrease in BBM fluidity play an important role in the impaired renal tubular Pi transport and adaptation to a low-Pi diet.

Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acyl (un)saturation to membrane fluidity measured by fluorescence polarization.

Steady-state fluorescence polarization (P) measurements, using the probe 1,6-diphenyl-1,3,5-hexatriene, in a large variety of well-defined liposomes at 25 degrees C allowed a quantitative description of the contributions of cholesterol, sphingomyelin, and (un)saturation of fatty acyl groups in the various phospholipids to the structural order (or the mutual affinity) of membrane lipids. The P values for liposomes prepared from lipid extracts of natural (purified) membranes of various origins could be more or less predicted (calculated) from the relative contributions of the individual lipid components. In all cases, the polarization varied with the cholesterol/phospholipid molar ratio (C/PL) according to the equation P = Pplat -(Pplat -Pzero) exp(-alpha C/PL), in which Pzero refers to the polarization without cholesterol and Pplat is a maximal plateau value, reached at a high C/PL (greater than 1). The "cholesterol-ordering coefficient" alpha of the phospholipids was found to increase with the fraction of sphingomyelin or dipalmitoylphosphatidylcholine molecules, indicating that the susceptibility of phospholipids to be ordered by cholesterol is increased by these compounds. Pzero increases curvilinearly with the fraction of either of these molecules. Pplat increases linearly with the fraction of saturated acyl chains for most phospholipids. Highly unsaturated fatty acyl chains (e.g., 20:4 and 22:6) strongly depress Pplat but not Pzero. The results suggest that such phospholipids are unlikely to associate with cholesterol and may thus create extremely fluid membrane domains. The disproportionation of cholesterol in the cell can be understood by the differing composition of the phospholipids in plasma membranes and endomembranes and their ordering susceptibility (affinity) toward cholesterol.

Steady-state fluorescence polarization data in membranes. Resolution into physical parameters by an extended Perrin equation for restricted rotation of fluorophores.

An extended Perrin equation is derived applicable to the restricted rotation of fluorophores. The equation results in a relation between time-resolved (r infinity) and steady-state fluorescence anisotropy (rs) data. This relation contains a parameter m, which expresses the difference between rotational diffusion in a lipid membrane and that in an isotropic reference oil having the same rs value. The relation is in agreement with rs, r infinity literature data for a variety of artificial and biological membranes labeled with various probes. Cholesterol and fatty acyl unsaturation affect the value of m, but temperature does not. The results indicate that, as far as fluorescence depolarization is concerned, either liposomes of saturated phospholipids without cholesterol or liposomes of unsaturated phospholipids containing cholesterol are good model systems for biological membranes. The accuracy of estimating order parameters or rotational diffusion constants from rs data is discussed. The formalism described here introduces a novel way of applying Arrhenius plots and allows for an unambiguous interpretation of rs data.

The effects of pressure and cholesterol on rotational motions of perylene in lipid bilayers.

Using steady-state fluorescence polarization measurements, an isothermal pressure-induced phase transition was observed in dimyristoyl-L-alpha-phosphatidylcholine multilamellar vesicles containing perylene. The temperature-to-pressure equivalence, dT/dP, estimated from the phase transition pressure, P1/2, is about 22 K X kbar-1, which is comparable to values determined from diphenylhexatriene polarization (Chong, P.L.-G. and Weber, G. (1983) Biochemistry 22, 5544-5550). In addition, we have employed a new method, introduced in this paper, to calculate the rate of in-plane rotation (Rip) and the rate of out-of-plane rotation (Rop) of perylene in lipid bilayers. The effects of pressure and cholesterol on the rotational rates of perylene in two lipid bilayer systems have been examined. They are 1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC) multilamellar vesicles (MLV) and 50 mol% cholesterol in POPC (MLV). Rop is smaller than Rip due to the fact that the out-of-plane rotation requires a larger volume change than the in-plane rotation. Cholesterol seems not to affect Rop significantly, but pressure causes a decrease in Rop by about a factor of three. In contrast, the effects of pressure and cholesterol on Rip are less straightforward. At 1 atm cholesterol increases Rip by a factor of about two. Similarly, in the absence of cholesterol 1.5 kbar pressure essentially triples Rip. However, if both cholesterol is added and pressure is applied, Rip decreases sharply. The possible interactions between cholesterol and perylene are discussed.

An explanation of the low lecithin/sphingomyelin ratio of amniotic fluid in early pregnancy.

We have attempted to determine why the lecithin/sphingomyelin (L/S) ratio of amniotic fluid is lower than 2 during an early stage of pregnancy. We found that, at 16 wk of gestation, long before the fetal lung secretes lecithin into the amniotic fluid, the L/S ratio was about 1. High-density lipoprotein isolated from the amniotic fluid also had such a low L/S ratio. The L/S ratios of the high-density lipoprotein from umbilical cord blood and maternal blood, however, were much higher, viz. 3.7 (+/- 0.25) and 6.4 (+/- 0.33), respectively. The increase coincided with a decrease in their fluorescence polarization. We suggest that the low L/S ratio of 16 wk amniotic fluid is caused by lipolysis of its lecithin, which is derived from fetal or maternal high-density lipoproteins.

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements.

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, rs, is resolved into a fast decaying or kinetic component, rf, and an infinitely slow decaying or static component, r infinity. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; r infinity is proportional to the square of the lipid order parameter. An empirical relation between rs and r infinity is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.