Free Dye Concentration Research Articles

Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume expanded mice

Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume expanded mice

The kinetics of YOYO-1 intercalation into single molecules of double-stranded DNA

The cyanine dye, YOYO-1, has frequently been used in single DNA molecule imaging work to stain double-stranded DNA as it fluoresces strongly when bound. The binding of YOYO-1 lengthens the DNA due to bis-intercalation. We have investigated the kinetics of binding, via this increase in DNA length, for single, hydrodynamically-stretched molecules of lambda DNA observed via Total Internal Reflection Fluorescence (TIRF) microscopy. The rate and degree of lengthening in 40mM NaHCO3 (pH 8.0) buffer depend upon the free dye concentration with the reaction taking several minutes to reach completion even in relatively high, 40nM, concentrations of YOYO-1. In the absence of overstretching of the DNA molecule, we determine the second order rate constant to be 3.8±0.7×105s−1M−1, the dissociation constant to be 12.1±3.4nM and the maximum DNA molecule extension to be 36±4%. The intercalation time constant (inverse of the pseudo-first order rate constant), τ, decreased from 309 to 62s as YOYO-1 levels increased from 10 to 40nM. The kinetics of binding help with interpretation of the behavior of DNA–YOYO-1 complexes when overstretched and establish defined conditions for the preparation of DNA–YOYO-1 complexes.

Characterizing The Equilibrium Blinking Behavior Of Fluorogens With Fluorogen Activating Proteins (FAPs)

We have recently developed FAPs (fluorogen activating proteins) that can specifically activate the fluorogenic dyes thiazole orange (TO) and malachite green (MG) with nanomolar affinities. Upon binding to FAPs, the otherwise dark fluorogens display thousands of fold increase in fluorescence intensity. The reversible interaction between the fluorogen and the FAP allows the same FAP to bind and activate fresh fluorogens in solution after one fluorogen dissociates from the FAP. Therefore, the binding and unbinding reactions lead to on-and-off (blinking) fluorescent signals at the single molecule level. As a result, this FAP-fluorogen system is ideal for generating a renewable, photostable signal and providing high photon flux. Since the localization accuracy is in principle limited by the number of photons collected from fluorescent probes, our module holds great promise for achieving super-resolution imaging (See abstract by Lidke, K.A. et al.). In this study, we characterize the blinking behavior of FAP-fluorogen pairs at equilibrium. We show that the on-time is highly dependent on the excitation power density and the off-time is controlled by the free dye concentration and therefore, is limited by diffusion. By adjusting the dye concentration in solution and the intensity of incident power, the on and off rates of the binding reaction are controllable and could be optimized to resolve the objects in time and space. Clones of the FAP with point mutations show different blinking behaviors, which suggests that these amino-acid residues could play an important role in FAP-fluorogen binding and indicates that structural differences of the FAP at the binding interface can change the photochemical and photophysical properties of fluorogen.

Diethylaminoethylation of Chitin and the Adsorption of Acid Dyes onto the Resulting Polymer

Water-soluble chitin derivatives have been synthesized by the diethylaminoethylation (DEAE) reaction and the adsorption characteristics of the resulting polymeric adsorbents towards two acid dyes (CI Acid Orange 7 and CI Acid Red 88) were studied. The adsorption behaviours towards these dyes were also compared with those exhibited by two water-insoluble commercially available derivatives. The conventional water-insoluble DEAE-chitins showed a high adsorptive capacity towards CI Acid Orange 7 compared to that on untreated chitin and alkali-treated chitin. A similar trend was also found for CI Acid Red 88 dye, where the increase in adsorption was characterized more clearly. However, the adsorption capacities towards both dyes exhibited by the water-soluble DEAE-chitins were increased significantly at a particular free dye concentration, with the adsorptive power also being increased both with the degree of DEAE substitution and the hydrophobicity of the dyes.

Interaction of the neuronal marker dye FM1-43 with lipid membranes: Thermodynamics and lipid ordering

The fluorescent dye FM1-43 labels nerve terminals in an activity-dependent fashion and has been found increasingly useful in exploring the exo- and endocytosis of synaptic vesicles and other cells by fluorescence methods. The dye distributes between the aqueous phase and the lipid membrane but the physical-chemical parameters characterizing the adsorption/partition equilibrium have not yet been determined. Fluorescence spectroscopy alone is not sufficient for a detailed elucidation of the adsorption mechanism since the method can be applied only in a rather narrow low-concentration window. In addition to fluorescence spectroscopy, we have therefore employed high sensitivity isothermal titration calorimetry (ITC) and deuterium magnetic resonance ( 2H-NMR). ITC allows the measurement of the adsorption isotherm up to 100 μM dye concentration whereas 2H-NMR provides information on the location of the dye with respect to the plane of the membrane. Dye adsorption/partition isotherms were measured for neutral and negatively-charged phospholipid vesicles. A non-linear dependence between the extent of adsorption and the free dye concentration was observed. Though the adsorption was mainly driven by the insertion of the non-polar part of the dye into the hydrophobic membrane interior, the adsorption equilibrium was further modulated by an electrostatic attraction/repulsion interaction of the cationic dye ( z=+2) with the membrane surface. The Gouy-Chapman theory was employed to separate electrostatic and hydrophobic effects. After correcting for electrostatic effects, the dye-membrane interaction could be described by a simple partition equilibrium ( X b= Kc dye) with a partition constant of 10 3–10 4 M −1, a partition enthalpy of Δ H=−2.0 kcal/mol and a free energy of binding of Δ G=−7.8 kcal/mol. The insertion of FM1-43 into lipid membranes at room temperature is thus an entropy-driven reaction following the classical hydrophobic effect. Deuterium nuclear magnetic resonance provided insight into the structural changes of the lipid bilayer induced by the insertion of FM1-43. The dye disturbed the packing of the fatty acyl chains and decreased the fatty acyl chain order. FM1-43 also induced a conformational change in the phosphocholine headgroup. The −P-N + dipole was parallel to the membrane surface in the absence of dye and was rotated with its positive end towards the water phase upon dye insertion. The extent of rotation was, however, much smaller than that induced by other cationic molecules of similar charge, suggesting an alignment of FM1-43 such that the POPC phosphate group is sandwiched by the two quaternary FM1-43 ammonium groups. In such an arrangement the two cationic charges counteract each other in a rotation of the −P-N + dipole.

Linearized Colorimetric Assay for Cremophor EL: Application to Pharmacokinetics after 1-Hour Paclitaxel Infusions

Cremophor EL (CrEL) is a polyoxyethylated castor oil surfactant used in the intravenous formulation of the anticancer drug paclitaxel (Taxol). Quantitative determination of CrEL in patient samples can be achieved by complexation of the compound with the Coomassie brilliant blue G-250 dye in protein-free extracts [Sparreboom, A., Loos, W. J., Verweij, J., De Vos, A. I., Van der Burg, M. E. L., Stoter, G., and Nooter, K.,Anal. Biochem.255, 171–175 (1998)]. A disadvantage of this method of CrEL determination is that the assay plot of absorbance at 595 nm, the peak wavelength of the CrEL–dye complex, versus the concentration of the surfactant is not linear. The present study shows that the nonlinearity is associated with a decrease in the free dye concentration and a reduction in complex formation by increasing the CrEL concentration. By measurement of the ratio of absorbances at the maxima of the red (450 nm) and blue charge forms (595 nm) of Coomassie brilliant blue G-250, a full-scale linear relationship can be obtained over the entire range studied (0.500 to 10.0 μL/mL). Validation data revealed that transformation of the detection procedure exhibits significantly improved specificity, accuracy(≤ 6.33% relative error), and precision (< 10.0%) compared to our previous assay. The modified method was successfully applied to the measurement of CrEL in plasma of 11 cancer patients treated with a 1-h infusion of paclitaxel.

Equilibrium binding of DAPI and 7-aminoactinomycin D to chromatin of cultured cells.

The binding of 4',6-diamidino-2-phenylindole (DAPI) and 7-aminoactinomycin D (7-AAMD) to chromatin of intact cultured fibroblast nuclei was studied by cytofluorometry. Staining was performed at equilibrium at varying dye concentrations. By using large volumes of dye solutions, the free dye concentration became approximately equal to total dye concentration, and the estimation of affinity and number of binding sites was performed by Scatchard analysis. Since all Scatchard plots showed a definite curvature, indicating more than one class of binding sites, a two component regression was performed, although only the highest affinity binding sites were determined. The observed affinity constants did not change substantially between different preparational procedures for any of the dyes. The number of DAPI binding sites increased about 30% after detergent extraction. No such increase was observed for 7-AAMD. Fixation in formaldehyde, instead of ethanol, after detergent extraction had no effect on DAPI binding, but decreased the number of 7-AAMD binding sites with about 50%. Extraction of basic proteins with HCl resulted in about 100% increase of the number of binding sites for both dyes. It should be feasible to use this technique as an assay for the identification of proteins or other nuclear components important for the maintenance of chromatin structure.

Acridine orange as a probe for measuring pH gradients across membranes: Mechanism and limitations

Acridine orange is an optical probe commonly used to monitor pH gradients across membranes. In the present study, the changes observed in the visible absorption spectrum of acridine orange during intravesicular acidification of oat root plasma membrane vesicles are shown to be identical with those obtained by increasing the free dye concentration, adding anions, or lowering the temperature, but different from those obtained on addition of biological membranes. It is therefore suggested that the absorbance changes observed during the formation of the pH gradient are simply due to accumulation of free dye inside the vesicles and subsequent dimerization, and not the result of dye-membrane interactions. The proportion of monomeric acridine orange that could undergo dimerization decreased with decreasing temperature. Furthermore, in a membrane-free system different anions induced the formation of dimer-excimer complexes to different degrees. During the formation of the pH gradient permeant anions present in the reaction medium follow the movement of protons into the vesicles, and the intravesicular accumulation of anions thereby amplifies acridine orange quenching, the degree of amplification being dependent on the anion species. Therefore, the use of acridine orange, and probably all metachromatic dyes, as probes for monitoring pH gradients is limited, since these probes neither reflect quantitatively the amount of H + pumped nor the effect of anions and temperature on transmembrane H + transport.

The interaction of the potential-sensitive molecular probe merocyanine 540 with phosphorylating beef heart submitochondrial particles under equilibrium and time-resolved conditions

The interaction of the potential-sensitive extrinsic molecular probe merocyanine 540 (M540) with phosphorylating submitochondrial particles has been investigated under equilibrium and time-resolved conditions. The addition of ATP to a M540-membrane suspension produces oligomycin and CCCP-sensitive spectral changes with absolute maxima near 490, 530, and 565 nm; a 1- to 2-nm red shift of the dye absorption spectrum is also evident in the longer-wavelength region of the spectrum. In fixed-wavelength work, the energy-dependent optical signals were increased by the addition of nigericin and NH 4Cl, and could be subsequently restored to the control level by valinomycin or KSCN, respectively. These observations suggest that M540 is specifically sensitive to the membrane-potential portion of the electrochemical gradient presumably present in the submitochondrial particle system in the presence of substrate. Binding analyses based on the Langmuir adsorption isotherm and the direct linear method indicate that the M540 dissociation constant is decreased by the presence of ATP with little or no change in the maximum number of binding sites. The M540 dissociation constant was markedly decreased when 0.1 m NaCl was present in the medium, suggesting that the association of this probe with the membrane may be subject to considerable surface charge repulsion. Results from the binding analyses indicate that the origin of the energy-dependent spectral changes may be an enhanced association of M540 with the submitochondrial particle membrane resulting from the transfer of dye from the aqueous phase to membrane-binding sites. The time course of the NADH-, ATP-, or succinate-induced signal developed slowly, on a time scale of tens of seconds, and follows a second-order rate law, suggesting that the rate-limiting step in the development of the ATP-induced M540 signal may be the transfer of dye from the aqueous phase to membranebinding sites. The enhanced passive binding of M540 to the submitochondrial particle membrane in the presence of NaCl reduces the concentration of free dye apparently available to redistribute to the membrane when substrate is present, with a concomitant reduction in the observed pseudo-first-order and the second-order rate constants. If the effective free dye concentration is estimated from binding data and used in the plot from which the latter rate constant is obtained, the value of this constant compares favorably with the obtained in the absence of the electrolyte. In a concentration range as large as 0 to 10 μ m, the presence of M540 in submitochondrial particles has no effect on the rate constants characterizing three energy-dependent processes: the ATP-induced cytochrome c oxidase soret band shift, and the oxidation of cytochrome 1 and the reduction of pyridine nucleotide, both via ATP-driven reversed electron transport. The failure of M540 to impede the rates at which these energy-intensive processes develop suggests that the M540 anion does not permeate the particle membrane in the time scale covered by these experiments, 0.3 s to some 15 min.

Cooperative interaction of carboxymethyl cellulose with C.I. basic red 18 in aqueous solution

Abstract The binding isotherms of C.I. Basic Red 18 with carboxy-methyl cellulose having various degrees of substitution have been determined in an acetate buffer at pH 4.2 and 30°C by a dynamic dialysis technique. The isotherms show a sigmoidal increase of binding with increasing free dye concentration in solution. The results strongly suggest a cooperative mode of binding. In the interpretation of the results, McGhee and von Hippel's theory considering cooperativity in binding was used. The intrinsic binding constant K, the number of consecutive polymer residues occupied by a single dye molecule n, and the cooperativity parameter were determined. A molecular mechanism of binding is proposed.

A New method of determining the extent of binding of an ionic dye to a polyelectrolyte in solution

A method of determining the free dye concentration in a dye/polyelectrolyte solution is described. The method utilizes the tendency of cationic dyes to adsorb to a cellulose dialysis membrane, and the nature of the membrane binding is studied by spectrophotometric methods. The binding of the cationic dye toluidine blue to the polyanion sodium carboxymethylcellulose in solution is used as an example. The proposed technique gives reliable estimates of free dye concentration over a wide range of polyanion and dye concentrations, both in the presence and absence of added simple electrolyte. The method possesses inherent advantages over estimates of free dye concentration obtained by absorption spectrophotometry and is more versatile than fluorimetry.

THE INFLUENCE OF QUATERNIZED POLY (VINYLPYRIDINE)S ON THE DIFFUSION OF 4-AMINOAZOBENZENE DERIVATIVES AND METHYL ORANGE HOMOLOGS IN NYLON 6

For nylon 6-acetate buffer solutions at pH 5.7 the diffusion coefficient, D and the equilibrium dye concentration, Cs of 4-aminoazobenzene (D-I) and 4-[bis (2-hydroxyethyl)amino] azobenzene (D-II) were determined at 35°C by permeation experiments.The solutions contained poly (N-butyl-4-vinylpyridinium bromide) (C4P4VP) (degree of quaternization, DQ=100%) or poly (N-octyl-2-vinylpyridinium bromide) (C8P2VP) (DQ=42.6%).With the addition of C4P4VP or C8P2VP in the dye bath, Cs decreases, but D does not change. By assuming a constant partition coefficient between the free dye concentration in the bath, Cf and Cs, the apparent binding constants, Kapp of the dyes to the polymers were estimated.The apparent diffusion coefficients, Dapp of methyl, ethyl and butyl orange (MO, EO and BO) in nylon 6 were determined at 35°C by means of desorption method using an acetate buffer solution containing C4P4VP or sodium poly(styrenesulufonate) (PSSNa) (degree of sulfonation, DS=100%). Dapp values of the dyes increase with the addition of C4P4VP and decrease with PSSNa. Such behaviors were discussed based on a theory of fixed charge membranes.

EFFECT OF SODIUM POLY(STYRENE SULFONATE) ON THE DYEING PROPERTIES OF DISPERSE DYES TO NYLON 6

The diffusion behavior of p-aminoazobenzene (Dye I) and 4-N, N-di-hydroxyethyl aminoazobenzene (Dye II) in nylon 6 as sorbed from aqueous and aqueous sodium poly(styrene sulfonate) (PSSNa) solutions was examined. The addition of PSSNa in the dye bath causes a marked decrease in the equilibrium dye concentration on nylon 6. It is found that the proportionality holds between the equilibrium dye concentration, Cs and the free dye concentration in the bath, Cf estimated from the solubility data. This indicates that the dye bound to PSSNa has no affinity to nylon 6. Further, the effect of rotation of the film roll on the equilibrium sorption of Dye I was examined.

The effect of bovine albumin on the permeability of frog mesenteric capillaries.

Two types of experiment have been carried out to investigate the effect of albumin upon the permeability of frog mesenteric capillaries to unbound molecules of T1824. In the first series of experiments a single capillary was perfused, via a micropipette, with a Ringer solution containing T1824‐albumin complex and an excess of unbound T1824. The same capillary was then perfused with a solution containing approximately the same free T1824 concentration but no albumin. Experiments were carried out on fourteen capillaries and it was found that the presence of albumin approximately doubled the time of first appearance of dye outside the capillaries. In a second series of experiments a capillary was perfused with a solution of T1824 and no albumin, and the time of first appearance of dye outside the capillary was measured densitometrically. The same capillary was then perfused a second time with a solution containing approximately the same free dye concentration in the presence of T1824‐albumin complex. As soon as the capillary had filled with dye, it was closed off at a point downstream from the site of cannulation and the pressure in the closed‐off segment raised by an amount equal to the osmotic pressure of the albumin. The time of first appearance of dye from the capillary was measured. Eleven experiments were carried out and it was found that the presence of albumin doubled the time of appearance of dye from the capillary. Nine control experiments revealed no difference in the time of appearance of dye leaving a capillary perfused with the same solution under conditions of free flow or from a blocked segment. It is concluded that albumin halves the permeability of the frog mesenteric capillaries to T1824.

The membrane structure studied with cationic dyes. 2. Aggregation, metachromatic effects and pK a shifts.

Three effects concerning the organic cations, the aggregation, the metachromasy and the pKα shift, have been studied in comparison at high dye concentrations, with natural and synthetic polyanions and with energized submitochondrial particles.The metachromatic effect has been obtained by increase of the free cationic dye concentration or by interaction of the dye with natural, synthetic polyanions and energized submitochondrial particles. The metachromatic effect is dependent on the pH of the medium, the presence of ionized acidic groups, and the ionic strength of the medium.The polyanion‐induced metachromasy requires the ionization of the acidic groups of the polyelectrolyte. The metachromasy with neutral red requires an acidic pH if induced by increase of the dye concentration or by chondroitin sulphuric acid, whereas it takes place also at alkaline pH if induced by polystyren sulphonic acid or by energized particles. It is inhibited by increase of ionic strength in the case of agar and chondroitin sulphuric acid, but not in the case of the energized particles or polystyrene sulphonic acid.Interaction of neutral red with polystyrene sulphonic acid or energized particles results also in a large apparent pKα shift, which represents the mechanism for obtaining the metachromatic effects at alkaline pH. The apparent pKα, as measured from the extinction of the alkaline band is: 6.0 at 400 μM neutral red, 6.7 at 20 μM neutral red, 7.0 with chondroitin sulphuric acid, 7.1 with the deenergized particles, 8.0 with polystyrene sulphonic acid and 8.1 with energized particles. From thermodynamic considerations it is suggested that the pKα shift requires a decrease of the activity coefficient of the dye following the formation of ion pairs with anionic groups of the membrane. The pKα shift may be taken as a tool for discriminating between a dyeinduced and a membrane‐induced metachromatic effect.A model is proposed for the energized membrane, based upon electrostatic and hydrophobic interactions of the cationic dyes with a layer of oriented nucleophilic sites in an environment of intermediate polarity.

PROTEINS IN MEAT AND EGG PRODUCTS DETERMINED BY DYE BINDING

SUMMARY— Under optimum conditions Acid Orange–12 will bind to the proteins of meat and eggs. The dye‐binding capacity is somewhat greater for these products than the previously reported values for wheat and milk products. A standard curve relating free dye concentration to mg of protein in the sample as determined by the Kjeldahl method can be readily prepared for each type of product. The method is rapid and useful for composition control in ground meats, eggs and prepared mixes. Cooking has little effect on the dye‐binding capacity but hydrolysis of the proteins to the proteose‐peptone stage reduces the dye bound. The coefficient of variation averaged about 3% of the protein content for the samples of meat and eggs tested.

Linear and circular dichroism studies of DNA-monoaminoacridine complexes

The circular dichroism spectra of the DNA complexes with 1-, 2-, 3-, 4- and 9-aminoacridine and with four monoaminobenzacridines have been measured over a range of DNA-phosphate to dye ratios and, in the case of the 9-aminobenzo[a]acridine complex, over the pH range 3.3–10 and at high and low ionic strength. The linear dichroism spectra of streaming solutions of the DNA-dye complexes have been obtained with light propagated parallel and perpendicular to the flow-direction. The relations between the fraction of dye bound and the free dye concentration have been determined by spectrophotometric and by polarographic methods. The degree of binding at physiological pH is found to correlate with the relative antibacterial and mutagenic activities of the monoaminoacridines. The spectroscopic results support a modified intercalation model for the DNA-acridine complex in which the bound acridine molecule is sandwiched between two adjacent bases attached to a common sugar phosphate chain, and they indicate that a reduction of pH produces an unwinding of the two strands of the DNA helix additional to that due to the intercalation of bound dye.

The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye.

Abstract The binding of the intercalating dye ethidium bromide to closed circular SV40 DNA causes an unwinding of the duplex structure and a simultaneous and quantitatively equivalent unwinding of the superhelices. The buoyant densities of both intact (I) and singly nicked (II) SV40 DNA's were measured as a function of free dye concentration, and the moles dye bound per mole nucleotide, ν, calculated from the buoyant density shifts. The binding isotherms were determined over a dye concentration range extending from 0 to 600 μg/ml. in 5.8 m -CsCl. At high dye concentrations all of the binding sites in II, but not in I, are saturated. At free dye concentrations less than 5.4 μg/ml., I has a greater affinity for dye than II. At a critical amount of dye bound, ν = νc, I and II have equal affinities, and at higher dye concentration I has a lower affinity than II. At ν = νc, there are 375 molecules of dye bound per molecule of SV40 DNA. The number of superhelical turns, τ, present in I was calculated at each dye concentration using Fuller & Waring's (1964) estimate of the angle of duplex unwinding per intercalation. With this estimate, we have calculated that the duplex has been unwound by 4500 ° at νc, and that SV40 DNA contains about −13 superhelical turns in concentrated salt solutions. The number of superhelical turns in SV40 DNA I has been evaluated as a function of the free dye concentration. The value of τ changes from −13 to zero to +45 as the free dye concentration is increased to 600 μg/ml. dye. The sedimentation coefficient of SV40 I in 1 m -NaCl decreases upon addition of dye at first slowly, and then rapidly, until the sedimentation coefficient becomes equal to that of II at the same dye concentration. At higher dye concentrations the sedimentation coefficient of I rises rapidly and appears to level off. The sedimentation coefficient of II decreases only slightly and linearly over the same dye concentration range. From the free dye concentration at the minimum and the value of the binding constant in 1 m -NaCl given by Le Pecq (1965), we calculate that there are −16.0 ± 3.5 superhelical turns in SV40 I DNA. Our binding results have allowed us to estimate the dependence of the sedimentation coefficient of SV40 I DNA upon the number of superhelical turns in the presence of dye.