Fluorescence Polarization Measurements Research Articles

Pigment organization in the photosynthetic apparatus of Roseiflexus castenholzii

The light-harvesting–reaction center (LHRC) complex from the chlorosome-lacking filamentous anoxygenic phototroph (FAP), Roseiflexus castenholzii ( R. castenholzii) was purified and characterized for overall pigment organization. The LHRC is a single complex that is comprised of light harvesting (LH) and reaction center (RC) polypeptides as well as an attached c-type cytochrome. The dominant carotenoid found in the LHRC is keto-γ-carotene, which transfers excitation to the long wavelength antenna band with 35% efficiency. Linear dichroism and fluorescence polarization measurements indicate that the long wavelength antenna pigments absorbing around 880 nm are perpendicular to the membrane plane, with the corresponding Q y transition dipoles in the plane of the membrane. The antenna pigments absorbing around 800 nm, as well as the bound carotenoid, are oriented at a large angle with respect to the membrane. The antenna pigments spectroscopically resemble the well-studied LH2 complex from purple bacteria, however the close association with the RC makes the light harvesting component of this complex functionally more like LH1.

A Polarized View Of DNA Under Tension

Polarized fluorescence measurements are valuable tools for determination of local structure and dynamics of biomolecules. In many applications, a great difficulty is to orient the biomolecules in a fixed and well-defined orientation, such that absolute measures of the directions of the absorption and emission dipole moments are attainable. Often alignment in flow or in a compressed gel is used, but this yields heterogeneity in the orientations. Here we present a novel and different approach: we use two optical tweezers to extend and align a piece of DNA and measure, at the same time, the polarization of the fluorescence of intercalating dye molecules (YOYO).

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.

Study Of The Drug/lipid Interactions Between Cephalosporin Antibiotics And Liposomes By Complementary Techniques

The cephalosporins are bactericidal with both gram-positive and gram-negative activity widely prescribed because of their broad spectrum. Even though most of these antibiotics appear to penetrate the outer membrane through porin proteins, we investigated, as a primary step, the interaction at the water-lipid interface. The information of the intrinsic property of these drugs in permeate or simply interact with phospholipid bilayers becomes of great importance for a better understanding of the functions of a lipid component in the membrane translocation. For that, the structural order of lipid membranes was investigated by measuring fluorescence polarization of membrane-bound fluorophores such as 1,6-phenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH) in the presence and absence of different cephalosporin generations as a function of temperature. Location studies have been also carried out using electron paramagnetic resonance (EPR) spectroscopy, valuable tool for collecting information on the dynamics of lipids and membrane structure. The results obtained suggest that the incorporation of these antibiotics into DMPC and DMPG bilayer does not significantly modify their transition temperature whereas perceptible changes in the cooperativity of the phospholipid transition are observed.

Microchip-based homogeneous immunoassay using fluorescence polarization spectroscopy

We have realized fluorescence polarization immunoassay (FPIA) on a microchip in about 1 minute. FPIA is a homogeneous competitive immunoassay which is based on measuring fluorescence polarization after competitive binding of an analyte and a tracer to an antibody. We constructed a microfluidic FPIA system composed of a newly designed microchip, a laser, a CCD camera and an optical microscope with two specially installed polarizers-one fixed and one rotatable. Theophylline, a typical small drug molecule, was used as a model analyte. Theophylline and fluorescence-labeled theophylline were introduced through different inlets and combined in a 100 microm-wide microchannel where anti-theophylline antibody was added. To optimize the microchip design for FPIA, we investigated the diffusion time of theophylline and the mixing time of theophylline and antibody in this channel, which were 6 s and 36 s, respectively. We successfully carried out a quantitative analysis of theophylline in serum near the therapeutic range in 65 s. In FPIA, a larger tracer-antibody complex emits more polarized fluorescence than the tracer, and therefore, by increasing the antigen concentration in a sample, more polarization relaxation is observed since the tracer-antibody complex concentration is decreased and the tracer concentration is increased. Tracer binding to an antibody is directly measured by spectroscopic techniques without any separation process.This microchip-based FPIA is very simple and rapid, unlike microchip-based heterogeneous immunoassay, because it does not require several processes such as washing and reflowing and immobilizing of antibodies or antigens in the channel. In the future, microchip-based FPIA should find frequent use for point-of-care testing in the clinical field, where conventional FPIA has been used for laboratory tests.

Mapping microscope object polarized emission to the back focal plane pattern.

The back focal plane (BFP) intensity pattern from a high-aperture objective separately maps far- and near-field emission from dipoles near a bare glass or metal-film-coated glass/aqueous interface. Total internal reflection (TIR) excitation of a fluorescent sample gave a BFP pattern interpreted in terms of fluorescent dipole orientation and distance from the interface. Theoretical consideration of this system led to identification of emission characteristics that remove a dipole orientation degeneracy in conventional microscope fluorescence polarization measurements. BFP pattern inspection removes the degeneracy. Alternatively, a BFP mask blocking a small fraction of emitted light in a standard imaging microscope prevents uniform collection of the BFP intensity and also eliminates the degeneracy. The BFP pattern from a single photoactivated photoactivatable green fluorescent protein (PAGFP) tagged myosin in a muscle fiber was observed despite the large background light from the highly concentrated myosin tagged with unphotoactivated PAGFP. This was accomplished by imaging the pattern from a nontelecentric plane, where most of the background intensity's pattern was translated laterally from the single-molecule object's pattern. TIR/BFP pattern imaging requires a simple alteration of the fluorescence microscope and is consistent with single-molecule imaging in a fluorophore dense three-dimensional object like a muscle fiber.

Conformation and Dynamics of a Rhodamine Probe Attached at Two Sites on a Protein: Implications for Molecular Structure Determination in situ

Replica exchange molecular dynamics (REMD) calculations were used to determine the conformation and dynamics of bifunctional rhodamine probes attached to pairs of cysteines in three model systems: (a) a polyalanine helix, (b) the isolated C helix (residues 53-66) of troponin C, and (c) the C helix of the N-terminal region (residues 1-90) of troponin C (sNTnC). In each case, and for both diastereoisomers of each probe-protein complex, the hydrophobic face of the probe is close to the protein surface, and its carboxylate group is highly solvated. The visible-range fluorescence dipole of the probe is approximately parallel to the line joining the two cysteine residues, as assumed in previous in situ fluorescence polarization studies. The independent rotational motion of the probe with respect to the protein on the nanosecond time scale is highly restricted, in agreement with data from fluorescence polarization and NMR relaxation studies. The detailed interaction of the probe with the protein surface depends on steric factors, electrostatic and hydrophobic interactions, hydrogen bonds, and hydration effects. The interaction is markedly different between diastereoisomers, and multiple preferred conformations exist for a single diasteroisomer. These results show that the combination of the hydrophobic xanthylium moiety of bifunctional rhodamine with the carboxylate substitution in its pendant phenyl ring causes the probe to be immobilized on the protein surface, while the two-site cysteine attachment defines the orientation of its fluorescence dipole. These features allow the orientation of protein components to be accurately determined in situ by polarized fluorescence measurements from bifunctional rhodamine probes.

Ultrafast resonance energy transfer from a site-specifically attached fluorescent chromophore reveals the folding of the N-terminal domain of CP29

The photosynthetic minor antenna complex CP29 of higher plants was singly mutated, overexpressed in Escherichia coli, selectively labeled with the fluorescent dye TAMRA at three positions in the N-terminal domain, and reconstituted with its natural pigments. Picosecond fluorescence experiments revealed rapid excitation energy transfer (∼20 ps) from TAMRA covalently attached to a cysteine at either position 4 or 97 (near the beginning and end of the N-terminal domain) to the chlorophylls in the hydrophobic part of the protein. This indicates that the N-terminus is folded back on the hydrophobic core. In 20% of the complexes, efficient transfer was lacking, indicating that the N-terminus can adopt different conformations. Time-resolved polarized fluorescence measurements demonstrate that the non-transferring conformations only allow restricted rotational motion of the dye molecule. When TAMRA was attached to a cysteine at position 40, the overall transfer efficiency was far lower, reflecting a larger distance to the hydrophobic region.

Resin‐Bound Aminofluorescein for C‐Terminal Labeling of Peptides: High‐Affinity Polarization Probes Binding to Polyproline‐Specific GYF Domains

A polymer support for the solid-phase synthesis of C-terminally labeled carboxylic acids has been developed. Fluorophore-labeled peptides were constructed directly on the amino group of resin-bound aminofluorescein. Fmoc-protected aminofluorescein was coupled onto tritylpolystyrene, and the free phenolic hydroxyl positions of the fluorescein were blocked with suitable protecting groups. The mode of attachment was analyzed and found to be selective for the phenoxy ether linkage. The conditions for peptide synthesis on the labeling resin were investigated, and a small library of C-terminally labeled peptides was prepared. The fluorescence quantum yields of C-terminally labeled peptides were determined and indicated the suitability of the compounds for imaging and binding experiments. The obtained peptides were therefore investigated as fluorescence polarization probes. Two different proline-rich binding domains of the GYF family-CD2BP2 and PERQ2-were targeted by peptides labeled either C- or N-terminally. Reversible binding constants were determined by fluorescence polarization measurements and were verified by competition experiments with the corresponding unlabeled peptide. As a second control, the binding constants were measured by NMR titration experiments, recording the HSQC NMR spectra of (15)N-labeled proteins in the presence of the peptide polarization probes. Ligands with higher affinities than all others known previously were identified for both GYF domains. The competition assay with the developed fluorescent probe has a high statistical reliability and can thus be used for screening of GYF domain inhibitors.

Photodynamic therapy with intratumoral administration of Lipid‐Based mTHPC in a model of breast cancer recurrence

Generalized skin sensitization is a main drawback of photodynamic therapy with systemic administration of photosensitizers. We have evaluated the potential use of an intratumoral injection of a liposomal formulation of mTHPC (Foslip) in a mouse model of local recurrence of breast cancer. Mice were directly injected into the tumor (IT) with 25 microl of a Foslip suspension (0.15 mg/ml) and illumination (652 nm, 20 J/cm(2)) was performed at different time points with pathological assessment after 48 hours. In a parallel mice series plasma samples were obtained at different endpoints after IT Foslip injection for HPLC analysis and the tumors were subjected in toto to macrofluorescence imaging. Fluorescence polarization measurements were conducted in vitro to estimate the rate of sensitizer redistribution from liposomes. Optimal, albeit partial, cure rates were obtained at 24 hours post-sensitizer and uninistration. Inhomogeneous and weak fluorescence was observed at early time points and became maximal at 24 hours. Plasma levels of mTHPC increased until 15 hours. Fluorescence polarization measurements showed a slow sensitizer transfer from liposomes to model membranes. The weak intratumoral fluorescence at early time points could be explained by concentration quenching within the liposomes as evidenced from fluorescence polarization studies. Progressive mTHPC redistribution from liposomes and its further incorporation into tumor tissue resulted in fluorescence build-up over time with a maximum at 24 hours post-injection. This correlates perfectly with the best therapeutic effect at this time point. The absence of total cure can be attributed to inhomogeneous photosensitizer distribution. mTHPC is reabsorbed into the blood stream but the total administered amount is much reduced as opposed to systemic administration so that repeated PDT sessions might be favorable in terms of side effects and tumor response.

Chain-Length Dependent Nematic Ordering of Conjugated Polymers in a Liquid Crystal Solvent

In this communication we demonstrate the dependence of the solute order parameter on the solute molecular weight for polymer solutes dissolved in liquid crystalline solvents. Using ensemble absorption polarization spectroscopy together with single molecule fluorescence polarization measurements, we have determined the order parameter of the conjugated polymer MEH-PPV (poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene]) in the liquid crystal 5CB (4-cyano-4-n-pentylbiphenyl) as a function of polymer chain length. Ensemble absorption polarization measurements agree well with results obtained by single molecule fluorescence polarization spectroscopy, indicating a large-scale ordering of the MEH-PPV solute in 5CB. These results demonstrate that the increasing number of defects for larger polymer weights inherently limits the alignment of the polymer solute.

Structural stability of green fluorescent proteins entrapped in polyelectrolyte nanocapsules

Molecules of a green fluorescent protein mutant, GFPmut2, have been immobilized in nanocapsules, assemblies of charged polyelectrolyte multilayers, with the aim to study the effect of protein-polyelectrolyte interactions on the protein stability against chemical denaturation. GFPmut2 proteins turn out to be stabilized and protected against the denaturating action of small chemical compounds. The nanocapsule protective effect on GFPmut2 is likely due to protein interactions with the negatively charged polymers, that induce an increase in the local rigidity of the protein nano-environment. This suggestion is supported by Fluorescence Polarization measurements on GFPmut2 proteins bound to the NC layers.

Insertional Inactivation of Branched-Chain α-Keto Acid Dehydrogenase in Staphylococcus aureus Leads to Decreased Branched-Chain Membrane Fatty Acid Content and Increased Susceptibility to Certain Stresses

Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain alpha-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12 degrees C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.

Stereospecific Interaction of Bupivacaine Enantiomers with Lipid Membranes

S(-)-Bupivacaine has the pharmacotoxicological advantage over its antipode and racemate. The interaction with lipid membranes was compared between S(-)-, R(+)- and racemic bupivacaine. The bupivacaine-induced changes in membrane property were determined by turbidity and fluorescence polarization measurements of membrane preparations to which bupivacaine stereoisomers of 1.0-5.0 mmol/L were applied. Liposomal membranes were made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine without or with cholesterol (5 to 15 mol%), and nerve cell model membranes of 55 mol% different phospholipids and 45 mol% cholesterol. The purity and hydrophobic interaction of bupivacaine were analyzed by reversed-phase high-performance liquid chromatography. Both S(-)- and R(+)-bupivacaine were not different in lowering the phase transition temperature of membrane 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. S(-)-, R(+)- and racemic bupivacaine disordered 100 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomal membranes, although the potency was indistinguishable between stereoisomers. By adding cholesterol to membranes, however, the membrane-disordering effects showed stereostructure-specificity that was enhanced with increasing the cholesterol content (0 to 15 mol%). The enantio-differentiating effects resulted from neither impurities in enantiomers nor hydrophobic interaction with phosphatidylcholine acyl chains. Bupivacaine disordered nerve cell model membranes with the potency being S(-)-enantiomer < racemate < R(+)-enantiomer, which resembled their relative stereopotency in nerve and cardiac channel inhibition. Membrane-disordering stereospecificity disappeared in the membranes without containing cholesterol. Bupivacaine stereostructure-specifically interacts with membranes containing cholesterol, which is consistent with the clinical features of S(-)-bupivacaine. Membrane cholesterol appears to increase the chirality of lipid bilayers and enable them to interact with S(-)-, racemic and R(+)-bupivacaine differently.

Molecular Chaperones and the Assembly of the Prion Ure2p in Vitro

The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94-354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3].

Accurate Determination of the Limiting Anisotropy of Rhodamine 101. Implications for Its Use as a Fluorescence Polarization Standard

The S1-S0 limiting anisotropy of a widely used fluorophore, rhodamine 101, is determined with unprecedented accuracy. From time-resolved and steady-state fluorescence measurements in several solvents, it is shown that the limiting anisotropy of rhodamine 101 is for all practical purposes equal to the theoretical one-photon fundamental anisotropy value of 2/5, both in rigid and in fluid media. This fact, along with the favorable chemical and photophysical properties of rhodamine 101, point to its use as a standard for fluorescence polarization measurements. It is also shown that if the excitation pulse can be considered a delta impulse with respect to the time scale of the anisotropy decay (but not necessarily to the time scale of the intensity decay), then no deconvolution procedure is needed for anisotropy decay analysis.

Membrane Activity-Guided Isolation of Antiproliferative and Antiplatelet Constituent from Evodiopanax Innovans

Bark of Evodiopanax innovans (Araliaceae) was subjected to membrane activity-guided extraction and chromatographic fractionation. The potency to interact with lipid membranes and change their fluidity was determined by measuring fluorescence polarization of liposomal and cell membranes. Plant preparations, including the MeOH extract and the specified fraction, reduced the fluidity of model biomembranes prepared with different phospholipids and cholesterol. Further purification led to the isolation of maltol 3- O-β-glucopyranoside, which inhibited tumor cell growth and platelet aggregation, together with rigidifying the cell membranes as well as the membrane-active antitumor compound (-)-epigallocatechin gallate and doxorubicin. The isolate at 100 μM and 1.0 mM showed growth inhibition of 13–49% against tumor cells cultured for 24 and 48 h. At 1.8–3.6 mM, it also produced 50% inhibition of platelet aggregation induced by collagen, adenosine 5′-diphosphate and thrombin. E. innovans is considered as a medicinal plant containing a potent bioactive constituent that exerts antiproliferative and antiplatelet effects through interaction with cell membranes to modify their fluidity.

From molecular interactions to tissue organization—Diversity of evolving cytometric approaches from a Hungarian perspective

From molecular interactions to tissue organization—Diversity of evolving cytometric approaches from a Hungarian perspective

NMR Studies of the Dynamics of a Bifunctional Rhodamine Probe Attached to Troponin C

Fluorescence polarization measurements of bifunctional rhodamine (BR) probes provide a powerful approach to determine the in situ orientation of proteins within ordered complexes such as muscle fibers. For accurate interpretation of fluorescence measurements, it is important to understand the probe dynamics relative to the protein to which it is attached. We previously determined the structure of the N-domain of chicken skeletal troponin C, BR-labeled on the C helix, in complex with the switch region of troponin I, and demonstrated that the probe does not perturb the structure or dynamics of the protein. In this study, the motion of the fluorescence label relative to the protein has been characterized using NMR relaxation measurements of 13C-labeled methyl groups on the BR probe and 15N-labeled backbone amides of the protein. Probe dynamics were monitored using off-resonance 13C-R(1rho), 13C-R(1) and {1H}-13C NOE at magnetic field strengths of 500, 600, and 800 MHz. Relaxation data were interpreted in terms of the overall rotational correlation time of the protein and a two-time scale model for internal motion of the BR methyl groups, using a numerical optimization with Monte Carlo parameter error estimation. The analysis yields a 1.5 +/- 0.4 ps correlation time for rotation around the three-fold methyl symmetry axis, and a 0.8 +/- 0.4 ns rotational correlation time for reorientation of the 13C-14N bond with an associated S2s of 0.79 +/- 0.03. Order parameters of the backbone NH vectors in the helix to which the probe is attached average S2 approximately 0.85, implying that the amplitude of independent reorientation of the BR probe is small in magnitude, consistent with results from fluorescence polarization measurements in reconstituted muscle fibers.

Mixed Monolayer and Micelle Formation of Cationic and Zwitterionic Surfactant of Identical Hydrocarbon Tail in an Aqueous Medium: Interfacial Tension, Fluorescence Probe, Dynamic Light Scattering, and Viscosity Studies

We have studied the properties of the mixed monolayers and the mixed micelles formed from the mixture of the cationic surfactant dodecyltrimethylammonium bromide (DTAB) and the zwitterionic surfactant dimethyldodecylammoniopropanesulfonate (DPS), that have the identical hydrocarbon tail but different polar head groups, by measuring interfacial tension, fluorescence, dynamic light scattering (DLS), and viscosity. From the results of interfacial tension measurements, the various interfacial and bulk parameters such as the maximum surface excess, the surface pressure at the critical micellar concentration, and the standard free energies of interfacial adsorption and of micellization have been evaluated. The deviation from the ideal of the mixed micelles has been discussed on the basis of Clint theory. The interaction parameters in the mixed micelles and in the mixed monolayers, and also the compositions of the mixed micelles and the mixed monolayers were obtained with the help of regular solution theory. From a static fluorescence quenching method, the mean micellar aggregation number of pure and mixed micelles was obtained. Microviscosity in the pure and mixed micelles was monitored by fluorescence polarization measurements using Rhodamine B (RB) as a fluorescence probe. The hydrodynamic radii of the micelles were obtained in the mixtures at different mole fractions of surfactant from DLS measurements. The change of viscosity with α of DTAB revealed a minimum, and it was shifted to a higher αvalue in the mixture of high concentration of surfactant. This minima suggested a structural change in the mixed micelles.