Early changes in concanavalin A-stimulated lymphocytes detected by the fluorescent probe N-phenyl-1-naphthylamine

Summary The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 g/ml concanavalin A (Con A) for 30 min at 37° C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by ³H-thymidine assay.Increased fluorescence was not observed in the presence of 10 mM -methyl mannoside, 5mM sodium azide, 10^–5 M cytochalasin B, or Ca²⁺-free culture medium.However, incubation with 10^–5 M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response.Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweed mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.Supported by a grant from the Anti-Cancer Council of VictoriaWe are grateful to Miss R. Jenkins and Mr. R. McGready for preparations of succinyl-Con A, to Dr. H.A. Ward for helpful discussion, and to Dr. M. Hohnes of the Walter and Eliza Hall Institute for providing BALB/c.nu mice     

A fluorimetric method for the estimation of the critical micelle concentration of surfactants

The present work investigates the possibility of a rapid estimation of critical micelle concentration (cmc) of surfactants by means of soluble fluorescent probes. The effect of nonionic or differently charged surfactants on the fluorescent properties of the anionic 8-anilino-1-naphtalenesulfonic acid magnesium salt (ANS) or cationic rhodamine 6G has been investigated. The possibility of cmc evaluation depends on the appropriate selection of the dye-detergent couple. ANS has to be used with anionic surfactants; on the other hand, rhodamine 6G has to be used with cationic detergents. Both ANS and rhodamine 6G have been proved to be effective with either zwitterionic or nonionic surfactants. Plots of ANS fluorescence increase or rhodamine 6G decrease vs surfactant concentration give two straight lines whose intersection indicates the cmc of the detergent. Under all these conditions the fluorescent probe does not interfere with the micellization process. Excitation of the fluorescent probes at the isosbestic point does not affect the evaluation of the cmc of the detergent. The method applies for linear or steroid surfactants and is independent of the cmc value within a wide range of concentrations.     

5-Dodecanoylaminofluorescein as a probe for the determination of critical micelle concentration of detergents using fluorescence anisotropy

A method for determining the critical micelle concentration (CMC) of various detergents based on fluorescence polarization (anisotropy) of the lipophilic probe 5-dodecanoylaminofluorescein is presented. Nonionic, cationic, anionic, and steroid-based detergents can all be evaluated by this method and the determined CMC values of selected detergents agree well with those reported in the literature. In addition, we report the CMC of domiphen bromide, whose CMC value has not previously been described. In the case of ionic detergents, the method described is particularly sensitive at discerning changes in the CMC with increasing ionic strength of the medium and can discriminate detergent CMCs in 5 mM versus 25 mM buffering components. The described fluorescence polarization technique allows very low (submicromolar) concentrations of probe to be employed, thus minimizing the perturbation of micelle formation by 5-dodecanoylaminofluorescein insertion.     

Temperature dependence of N-phenyl-1-naphthylamine binding in egg lecithin vesicles

The temperature dependence of the binding of PhNapNH₂ ($\text{N-}\text{phenyl-1-naphthylamine}$) to vesicles of egg phosphatidylcholine has been determined. The Arrhenius plot of the association constant exhibits a discontinuity at 20.9 °C, some 30 °C above the broad phase transition region of the phospholipid. In the temperature range above 20 °C, $\text{ΔH}{}^0\text{= ?6100}\text{cal·mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 9.7}\text{e. u.}$; in the temperature range below 20 °C, $\text{ΔH}{}^0\text{= 0}\text{cal · mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 30.4}\text{e. u.}$ These values are consistent with the view that there are well ordered lipid-lipid bonds below 20 °C which are significantly less important above this temperature. The order in the temperature range of 5 to 20 °C, though significantly greater than that above 20 °C, is still significantly less than that in the crystalline state.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic probe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, D-lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Determination of surfactant critical micelle concentration by a novel fluorescence depolarization technique

A novel method using fluorescence depolarization to determine the critical micelle concentrations (CMC) of surfactants was developed. Fluorescence anisotropies of Triton X-100, sodium dodecyl sulfate, and sodium cholate were measured using 1,6-diphenyl-1,3,5-hexatriene as a fluorescence probe. Fluorescence anisotropy decreased with increasing surfactant concentrations below the CMC and leveled off above the CMC. The depolarization method does not depend on the concentration of DPH and is largely immune to light-scattering problems encountered in turbid aqueous systems.     

Multiwavelength method for measuring concentration of free cytosolic calcium using the fluorescent probe indo-1

It is assumed that the spectra of fluorescent probes indo-1 and fura-2 in the cytoplasm are linear combinations of the spectra of calcium-bound and free probes with weight factors proportional to the concentrations of these forms. When the concentration of calcium is measured by the dual-wavelength method, the above assumption is employed without testing. A multiwavelength method for measuring free cytosolic calcium concentration is described in the present study. The method is based on the registration of the fluorescence spectra of the probe with an optical multichannel analyzer and deconvolution of the spectra into components, corresponding to free and bound forms of the probe. A mismatch is also calculated to allow estimation of deconvolution accuracy. It was found that the spectra, recorded in aqueous calibration solution with varying calcium concentrations, can be deconvoluted into components, obtained both in the absence of calcium and at its saturating concentration. When the spectrum of the probe in the cytoplasm is deconvoluted into the same components the mismatch is higher. When aqueous calibration is used, the cytosolic calcium concentration determined by the dual-wavelength method is dependent considerably on the selected wavelengths. Our data indicate that this phenomenon may be associated with the lower polarity of cytoplasm compared to the aqueous calibration solution. Addition of either ethanol or glycerol into the calibration medium results in a considerable decrease in the mismatch. The optimal concentration of ethanol is 22-32%, and depends on the type and condition of cells tested. It is shown that the use of calibration spectra obtained in aqueous solutions leads to considerable overestimation of cytosolic calcium concentration.     

High-throughput evaluation of the critical micelle concentration of detergents

Determination of the critical micelle concentration (CMC) value of detergents routinely used in biological applications is necessary to follow possible changes due to different buffer compositions (e.g., temperature, pH) such as those in solutions that are used for protein activity assays or crystallization. Here we report a method to determine the CMC values of detergents through a fast and robust assay that relies on the fluorescence of Hoechst 33342 using a 96-well plate reader. Furthermore, this assay provides the possibility and sensitivity to measure the CMC of detergent mixtures. The examples described here emphasize the potential and applicability of this assay and demonstrate that analysis of the physicochemical parameters of detergents can now be investigated in virtually every laboratory.     

The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of purified Escherichia coli cytochrome o incorporated into phospholipid vesicles

N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to the redox state of purified cytochrome o incorporated into lipid vesicles formed from purified or E. coli phospholipids. NPN was bound to the proteoliposomes to produce a steady-state level of fluorescence intensity. Addition of the substrate ascorbate, in the presence of phenazine methosulfate as an electron donor, did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of the substrate by molecular oxygen catalyzed by cytochrome o, there was an increase in the fluorescence of NPN. This coincided with the reduction of cytochrome o. Reoxidation of the cytochrome by addition of oxygen decreased the fluorescence to steady-state levels until the oxidant had been completely reduced. The fluorescence changes were dependent on the incorporation of cytochrome o into phospholipid vesicles but were insensitive to the state of energization of the vesicle membrane.     

Interfacial properties and critical micelle concentration of lysophospholipids

The critical micelle concentration (cmc) of several lysophospholipids and of a lysophospholipid analogue was determined from surface tension measurements using the maximum bubble pressure method and/or 31P NMR. The use of the maximum bubble pressure method has now been extended to micromolar concentrations of surfactant, and the experimental parameters that effect its use have been explored. Surface activity was found to vary with changes in the chain length and in the headgroup polarity of the lysophospholipid. The cmc's for 1-decanoyl-, 1-dodecanoyl-, 1-tetradecanoyl-, and 1-hexadecanoyl-sn-glycero-3-phosphocholine are 7.0, 0.70, 0.070, and 0.007 mM, respectively. The cmc's for 1-decanoyl- and 1-dodecanoyl-sn-glycero-3-phosphoethanolamine are 4.4 and 0.33 mM, respectively. The cmc for dodecylphosphocholine, a lysophospholipid analogue, was found to be 1.1 mM. The cmc's for 1-tetradecanoyl- and 1-hexadecanoyl-sn-glycero-3-phosphoglycerol were found to be 3.0 and 0.60 mM, respectively, in pure water. In 0.1 M Tris-HCl (pH = 8.0), their cmc's are 0.16 and 0.018 mM, respectively. Surface tension and adsorption density values determined at the cmc are reported for each compound. The relationship of dynamic surface tension and lipid purity is discussed. These studies provide information about the micellization and interfacial properties of several biologically important lysophospholipids.     

Virucidal activity of cetyltrimethylammonium bromide below the critical micelle concentration

Abstract The virucidal activity of cetyltrimethylammonium bromide (CTAB) was investigated against a variety of different lipid-containing and non-lipid-containing bacterial viruses and 2 mammalian viruses. In all cases, the maximum virucidal effectiveness was obtained under conditions of low ionic strength and moderately basic pH. The virucidal effect is present well below the critical micelle concentration of CTAB, indicating that the initial interaction with viral surfaces is by monomers of CTAB and sodium dodecyl sulfate (SDS) exhibited no virucidal activity.     

Rapid determination of surfactant critical micelle concentration in aqueous solutions using fiber-optic refractive index sensing

We describe a simple and rapid method for determining the critical micelle concentration (CMC) of surfactants from fiber-optic measurements of refractive index. The refractive index of an aqueous surfactant solution was monitored as the surfactant concentration was increased using an automated dispensing system. On reaching the surfactant’s CMC value, an abrupt change was observed in the rate of increase of the refractive index with increasing concentration. The measurement system provides rapid semiautomatic data collection and analysis, increasing the precision, sensitivity, and range of applicability of the technique while substantially decreasing the amount of manual intervention required. Measurements of CMC for sodium dodecyl sulfate (8.10 mM), cetyltrimethylammonium chloride (1.58 mM), and Triton X-100 (0.21 mM) were in excellent agreement with values previously reported in the literature. The method is applicable to cationic, anionic, and nonionic surfactants, and it offers a facile, in situ, and sensitive means of detecting micelle formation over a broad range of CMC values larger than 10⁻¹ mM.     

Determination of cysteine on low-density lipoproteins using the fluorescent probe, 5-iodoacetamidofluoresceine

Using the fluorescent sulfhydryl probe, 5-iodoacetamidofluoresceine, to label the free sulfhydryl of low-density lipoprotein, the positions of two cysteine residues in apolipoprotein B were located. The tryptic peptides containing the fluorescent probe were isolated by high-performance liquid chromatography systems and sequenced by automatic techniques. The free cysteine residues of apolipoprotein B-100 on low-density lipoprotein are located at positions 3734 and 4190, either or both of which can potentially form a disulfide linkage with apolipoprotein(a) in lipoprotein(a).     

Label-free critical micelle concentration determination of bacterial quorum sensing molecules

A practical label-free method for the rapid determination of small-molecule critical micelle concentration (CMC) using a fixed-angle light-scattering technique is described. Change in 90° light scattering at a fixed wavelength of incident radiation with increasing bacterial quorum molecule concentration and the observation of a break point is used to determine CMC. In our study, this technique is utilized to investigate the aqueous CMC of previously uncharacterized Pseudomonas aeruginosa quorum sensing signaling molecules (QSSM) belonging to the n-acylhomoserine lactone and 2-alkyl-4-quinolone classes. Several were found to form micelles within a physiologically relevant concentration range and potential roles of these micelles as QSSM transporters are discussed. The influence of temperature and the presence of biological membranes or serum proteins on QSSM CMC are also investigated and evidence is obtained to suggest the QSSMs studied are capable of both membrane and serum protein interaction. This demonstrates that the fixed-angle light-scattering technique outlined can be used simply and rapidly to determine small-molecule CMC under a variety of conditions.     

Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge

Upon mixing detergent solutions with the neutral fluorescent molecule 1,6-diphenyl-1,3,5-hexatriene a large increase in fluorescence is observed if detergent exceeds the critical micelle concentration. This property has been used to determine the critical micelle concentration of anionic, uncharged, zwitterionic, and cationic detergents. Regardless of detergent charge, the critical micelle concentrations obtained agree with the values obtained by other methods. This fluorescence assay is both sensitive and rapid, and should provide a simple and general method for determination of critical micelle concentration of any detergent.     

Spin label study of detergents in the region of critical micelle concentration.

A series of spin probes was employed to examine the behavior of the detergent sodium dodecyl sulfate (SDS) at concentrations above and below the critical micelle concentration (cmc). The existence of detergent aggregates below the cmc was evidenced by the appearance of composite electron spin resonance (ESR) spectra for probes that have measurable solubility in water. The spectra were indicative of two probe populations: one in an aqueous environment and another in detergent aggregates. The ESR spectra of probes which are highly insoluble in water exhibited line broadening due to intermolecular spin exchange interactions, indicating that the probes were concentrated in detergent aggregates present below the experimental cmc. The results are discussed in terms of their significance for the study of the mechanisms of micelle formation and for the detection of detergent aggregates at very low concentrations.     

Noninvasive methods to determine the critical micelle concentration of some bile acid salts

In this work the critical micelle concentrations (cmc) of four bile salts, sodium cholate, sodium glycocholate, sodium deoxycholate, and sodium glycodeoxycholate, are determined and presented. Three independent noninvasive methodologies (potentiometry, derivative spectrophotometry, and light scattering) were used for cmc determination, at 25 degrees C with ionic strength adjusted to 0.10 M with NaCl. Spectrophotometric and potentiometric studies of some bile salts were also executed at various ionic strength values, thus allowing the influence of the ionic strength on the cmc value of the bile salt to be assessed. A critical comparison of the cmc values obtained with data collected from the literature is presented. Furthermore, this work makes an evaluation of the conceptual bases of different methodologies commonly used for cmc determination, since variations in the results obtained can be related mainly to different intrinsic features of the methods used (such as sensitivity or the need to include tracers or probes) or to the operational cmc definition applied. The undoubted definition of the experimental bile salt concentration that corresponds to cmc (operational cmc) is essential since in the case of these amphiphiles the formation of micelles is not as abrupt as in the case of ordinary association colloids. The biphasic nature of their aggregation leads to a "round-shaped" variation of the experimental parameters under analysis, which makes difficult the evaluation of the cmc values and can be responsible for the different results obtained.     

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.