Model of detergent-induced spectral changes of the B800-850 complex from Chromatium minutissimum

The absorption and circular dichroism spectra of the B800-850 complex from Chromatium minutissimum before and after the Triton X-100 treatment were simulated by means of standard exciton theory, taking into account inhomogeneous broadening. To explain the spectral changes of the B800-850 complex treated with Triton X-100, we have assumed that all bacteriochlorophyll pigments absorbing at 850 nm exhibit the same additional rotation of approximately 20 degrees around the axis perpendicular to the membrane plane. This has been sufficient to fit the transformation in absorption and circular dichroism spectra induced by detergent treatment of the B800-850 complex.     

Surfactant micelles containing protoporphyrin IX as models of primitive photocatalytic systems: a spectral study

Micelles of various surfactants containing protoporphyrin IX (PPIX) have been studied as models of primitive membrane-like photocatalytic systems. Spectral characteristics (absorption spectra, fluorescence emission and excitation spectra, fluorescence quantum yields and lifetimes) have been measured to indicate specific interactions of PPIX molecules with the micelles. Two types of PPIX aggregates are probably formed in water: non-fluorescent clusters corresponding to the absorption peak at 642-648 nm and fluorescent friable dimers with strong solute-solvent interactions corresponding to the absorption peak at 618 nm. The aggregates are solubilized by the micelles, which results in an increase of the fluorescence quantum yield (and thus in the increase of the PPIX sensitizing ability). Solubilization of PPIX molecules by SDS-micelles is enhanced by the partial neutralization of the negative surface charge of the micelles. Neutral Triton X-100 micelles solubilize PPIX much better than SDS particles; however, some of the clusters formed in the bulk aqueous phase of the detergent-water system remain aggregated in the crown of the micelle. The positively charged CTAB micelles have been shown to provide the best solubilization of PPIX accompanied by the highest increase of its emitting activity. The results are discussed in terms of the possible role of PPIX-containing membraneous systems in primitive photosynthesis.     

Spectroscopic properties of protochlorophyll forms in Triton X-100 detergent micelles

Protochlorophyll (PChl) forms were performed in Triton X-100 detergent micelles. The concentration of Triton X-100 was 7·10^?4 M (above the critical micellar concentration); the concentration of PChl varied between 1.6·10^?5 and 1.8·10^?4 M. Absorption, fluorescence and circular dichroism (CD) spectra were registered. The absorption spectra were resolved into Gaussian components by computer analysis. PChl forms with absorption bands at 632–634, 638, 652–654, 663–664, 668 and 676 nm and with fluorescence emission bands at 634–636, 640–644, 652–655, 677–678, 686 and 694–696 nm were observed in micellar solutions of different PChl concentrations. The CD spectra showed a strong dependence on the concentration of PChl: positive CD signals or positive Cotton effects were observed in the vicinity of 650 nm. The intensity of these signals increased in parallel with increasing concentration of PChl. No CD signals were found in the region of the longer wavelength absorption bands. These data show that the PChl exists in many different forms in this system, and the spectroscopic properties of these forms are determined by different molecular interactions viz., interactions of PChl with Triton X-100 or water molecules and/or by the aggregation of PChl.     

Effect of phospholipids,Triton X-100 and biological membranes on redox systems involving tetrazolium salt reduction. Its implications for the assay of enzymatic activities

The influence of phospholipids and Triton X-100 on the time course of chemical and enzyme-mediated reductions of a commonly used tetrazolium salt, MTT, was studied. MTT reduction was followed by the absorbance changes at 570 nm. With ascorbate as reducing agent, a 3-fold increase in the initial rates of the absorbance changes and a 24 % increase in the final absorbance values were observed in the presence of Triton X-100 micelles or phospholipid vesicles. The enzyme-mediated reduction of MTT with NADH generated by the NAD-dependent lactate dehydrogenase was also enhanced in the presence of Triton X-100, phospholipids or erythrocyte membranes. No enhancement was observed following the enzymatic generation of NADH at 340 nm in the absence of MTT. The above findings were interpreted as arising from: a) solubilization or reduced MTT in the detergent micelles or phospholipid vesicles which favors the redox reaction occurring in the aqueous fase, and b) changes in the spectral properties of reduced MTT in aqueous and lipid-like media.     

Molecular organization of pheophytin a in aqueous solutions of detergents

Absorption, fluorescence and excitation fluorescence spectra of pheophytin a have been measured in aqueous solutions of nonionic (Triton X-100), anionic (sodium lauryl sulphate) and cationic (Cetyl pyridinium chloride) detergents at different concentrations and pH after system relaxation. By measuring the second derivative and differential spectra, it has been shown, that if detergent concentrations are lower than critical micelles concentration, or if the detergent is completely absent, the pigment forms conglomerates containing both dimeric and monomeric forms with an efficient energy transfer between them. If detergent concentrations are higher than critical micelles concentration, pheophytin a localizes in detergent micelle in monomeric form at neutral and acidic pH, and allomerizes at alkaline pH. The spectral characteristics of pheophytin a dimers in the conglomerate and its monomers in micelles poorly (if at all) depend on the sign of the detergent molecule charge.     

Energy transfer by chlorophyll beta in detergent micelles.

The concentration-dependent depolarization, concentration-dependent quenching, absorption and fluorescence spectra in solutions of chlorophyll beta-containing detergent micelles with Triton X-100 were studied in a concentration range of c equal to 0.4 muM-0.6mM chlorophyll beta and cd equal to 0.4-7.0 mM Triton X-100. The concentration-dependent depolarization obeys F?rster's theory of depolarization of fluorescence with a transfer distance parameter R0 equal to 43 plus or minus 2 A. The concentration-dependent quenching is described by an empirical formula for the relative fluorescence yield n/n0 equal to 1/[1+(c/c1/2)-2] given by Kelly and Porter (Kelly A. R. and Porter, G. (1970) Proc. R. Soc. Lond. Ser. A. 315, 149-161). With increasing chlorophyll beta concentration the red absorption band at 650 nm is shifted toward a longer wavelength and its width increases by 10nm, the intensity of the long wave fluorescence band increases about 720 nm. The results analysed in terms of these findings lead to the conclusions that chlorophyll beta molecules are (a) locally concentrated in the micelles up to the concentration range of in vivo conditions, (b) partly in an aggregated state capable for fluorescence, (c) the chlorophyll beta yields chlorophyll beta homotransfer may be about 3-26% of the homotransfer chlorophyll alpha yields chlorophyll-alpha depending on the ratio of their concentrations.     

Spectral properties of adrenaline in micellar environment

An absorption and fluorescence spectral and temporal studies on the solubilzation properties of adrenaline in micellar environment in sodium dodecyl sulfate (SDS) and in tetradodecyltrimethyl ammonium bromide (TTABr) has been carried out. Observed Stokes shifts have been correlated with polarity parameters which allowed an estimate of the dielectric constant of the adrenaline environment in SDS and TTABr micelles at 44 and 58, respectively. Experiments with methanol-water mixtures indicate that the hydrogen bonding formation with solvent and the hydrophilic nature of adrenaline influence its solubilization in micelles. Fluorescence and anisotropy decay analysis has shown that neutral adrenaline in SDS micelle is partitioned between aqueous phase (70%) and less polar, micellar phase (30%) and the interactions are limited to the Guy-Chapman layer without deeper penetration into micellar hydrophobic core.     

GM1-ganglioside-triton X-100 mixed micelles: Changes of micellar properties studied by laser-light scattering and enzymatic methods

The micellar properties of mixtures of GM1 ganglioside and the non-ionic amphiphile Triton X-100 in 25 mM Na phosphate-5 mM di Na EDTA buffer (pH = 7.0) were investigated by quasielastic light scattering in a wide range of Triton/GM1 molar ratios and in the temperature range 15–37°C. These measurements: (a) provided evidence for the formation of mixed micelles; (b) allowed the determination of such parameters as the molecular weight and the hydrodynamic radius of the mixed micelles; (c) showed the occurrence of statistical aggregates of micelles with increasing temperature and micelle concentration. Galactose oxidase was chosen for studying the relation between enzyme activity and micellar properties. The action of the enzyme on GM1 was found to be strongly dependent on the micellar structure. In particular: (a) galactose oxidase acted very poorly on homogeneous GM1 micelles, while affecting mixed GM1/Triton X-100 micelles; (b) at fixed GM1 concentration the oxidation rate increased by enhancing Triton X-100 concentration and followed a biphasic kinetics with a break at a certain Triton X-100 concentration; (c) the formation of statistical micelle aggregates was followed by inhibition of the enzyme activity.     

The Influence of Surfactants on Chlorophyll Binding Status and Excitation Energy Transfer in Photosystem ￠? of Wheat (in English)

Surfactants are widely used in the purification and research of structure and function of the protein complexes in photosynthetic membrane. To elucidate the mechanism of interaction between surfactants and photosystem Ⅰ (PSⅠ), effects of two typical surfactants, Triton X-100 and sodium dodecyl sulfate (SDS) on PSⅠ, were studied at different concentrations. The results were: SDS led to the reduction of apparent absorption intensity and blue shift of absorption peaks; while Triton X-100 led to the decrease of apparent absorption intensity in red region and blue shift of the peak, but to the increase of apparent absorption intensity in blue region. The fourth derivative spectra show that the longwavelength (669 nm and 683 nm) absorbing chlorophyll a was affected greatly and their relative changes of absorbance were axially symmetrical. The presence of surfactant could make the long wavelength fluorescence emission decrease greatly and a new fluorescence peak appeared around 680 nm, it was obvious that the surfactant interceded the transfer of excitation energy from antenna pigments to reaction center. The surfactants might affect the microenvironment of proteins, even the structure of PSⅠ protein subunits and hence changed the binding status of pigments with protein subunits, or the pigments might be released from the subunits. All of these might affect the absorption and the transfer of excitation energy.     

Energy transfer by chlorophyllb in detergent micelles

The concentration-dependent depolarization, concentration-dependent quenching, absorption and fluorescence spectra in solutions of chlorophyllb-containing detergent micelles with Triton X-100 were studied in a concentration range ofc = 0.4 μM–0.6mM chlorophyllb andc_d = 0.4–7.0mM Triton X-100. The concentration-dependent depolarization obeys Fo¨rster's theory of depolarization of fluorescence with a transfer distance parameterR₀ = 43 ± 2A?. The concentration-dependent quenching is described by an empirical formula for the relative fluorescence yieldη/η₀=sol1[1 + (cc_1/2)²] given by Kelly and Porter (Kelly A. R. and Porter, G. (1970) Proc. R. Soc. Lond. Ser. A. 315, 149–161). With increasing chlorophyll b concentration the red absorption band at 650 nm is shifted toward a longer wavelength and its width increases by 10 nm, the intensity of the long wave fluorescence band increases about 720 nm. The results analysed in terms of these findings lead to the conclusions that chlorophyllb molecules are (a) locally concentrated in the micelles up to the concentration range of in vivo conditions, (b) partly in an aggregated state capable for fluorescence, (c) the chlorophyllb →chlorophyllb homotransfer may be about 3–26 % of the homotransfer chlorophylla →chlorophyll-a depending on the ratio of their concentrations.     

Photodynamic effect in lysozyme: a kinetic study in different micellar media.

The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X-100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X-100 and SDS micelles was quantified by determining the respective associations constant (K(Lyso)). Values were 37 M(-1) for Triton X-100 and 514 M(-1) for SDS, indicating that the Lyso molecule binds Triton X-100 micelles effectively and SDS micelles even more strongly. Time-resolved phosphorescence detection (TRPD) indicates that the protein interacts with O2 (1deltag), with overall rate constants of the order of 10(8) M(-1)/S in direct micelles and 10(7) M(-1)/S in reverse micelles. Apparent reactive rate constants for eosin-sensitized photo-oxidation (singlet molecular oxygen [O2 (1deltag)]-mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O2 (1deltag) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo-oxidative reaction in SDS and in Triton X-100 at surfactant concentrations < 1 x 10(-2) M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was approximately 1 x 10(-5), the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O2 (1deltag).     

Properties of partially purified photosynthetic reaction centers from scenedesmus mutant 6E and Anabaena variabilis grown in the presence of diphenylamine

When membrane fragments of Anabaena variabilis grown in the presence of diphenylamine (designated diphenylamine-Anabaena) are treated with Triton X-100 and subjected to sucrose density gradient centrifugation, a bluish-green membrane fragment enirched in P700 is obtained. This high-P700 fragment, denoted HP700, contains three P700 molecules per 100 chlorophyll a molecules and reduces NADP at a rate that is approximately nine times higher than that of HP700 fragments prepared from normally cultured Anabaena by the use of Triton X-100 following extraction with organic solvents. An HP700 fragment has also been isolated from a carotenoidless Scenedesmus mutant 6E, by the use of Triton X-100 and sucrose density gradient centrifugation.

Both HP700 fragments show the characteristic rapid absorbance changes of P700 upon illumination. The fluorescence properties of the HP700 fragments at −196° are different from those of the original membrane fragments. At −196° the long wavelength fluorescence peak is located at a shorter wavelength (724 mμ) in the diphenylamine-Anabaena HP700 fragment and is lower in intensity than that observed with the membrane fragment. Long wavelength fluorescence at −196° is low in the flurorescence spectra of the membrane fragments of Scenedesmus mutant 6E and is barely observable in the HP700 fragment. The fluorescence spectra of the HP700 fragments of both diphenylamine-Anabaena and Scenedesmus mutant 6E at −196° show a shoulder or peak at 700 nm. The data on fluorescence properties of the HP700 fragments suggest that 730 nm fluorescence does not originate from P700.     

Triplet and fluorescing states of the CP47 antenna complex of photosystem II studied as a function of temperature.

Fluorescence emission and triplet-minus-singlet (T-S) absorption difference spectra of the CP47 core antenna complex of photosystem II were measured as a function of temperature and compared to those of chlorophyll a in Triton X-100. Two spectral species were found in the chlorophyll T-S spectra of CP47, which may arise from a difference in ligation of the pigments or from an additional hydrogen bond, similar to what has been found for Chl molecules in a variety of solvents. The T-S spectra show that the lowest lying state in CP47 is at approximately 685 nm and gives rise to fluorescence at 690 nm at 4 K. The fluorescence quantum yield is 0.11 +/- 0.03 at 4 K, the chlorophyll triplet yield is 0.16 +/- 0.03. Carotenoid triplets are formed efficiently at 4 K through triplet transfer from chlorophyll with a yield of 0.15 +/- 0.02. The major decay channel of the lowest excited state in CP47 is internal conversion, with a quantum yield of about 0.58. Increase of the temperature results in a broadening and blue shift of the spectra due to the equilibration of the excitation over the antenna pigments. Upon increasing the temperature, a decrease of the fluorescence and triplet yields is observed to, at 270 K, a value of about 55% of the low temperature value. This decrease is significantly larger than of chlorophyll a in Triton X-100. Although the coupling to low-frequency phonon or vibration modes of the pigments is probably intermediate in CP47, the temperature dependence of the triplet and fluorescence quantum yield can be modeled using the energy gap law in the strong coupling limit of Englman and Jortner (1970. J. Mol. Phys. 18:145-164) for non-radiative decays. This yields for CP47 an average frequency of the promoting/accepting modes of 350 cm-1 with an activation energy of 650 cm-1 for internal conversion and activationless intersystem crossing to the triplet state through a promoting mode with a frequency of 180 cm-1. For chlorophyll a in Triton X-100 the average frequency of the promoting modes for non-radiative decay is very similar, but the activation energy (300 cm-1) is significantly smaller.     

New chlorophyll-b forms in a chlorophyll-detergent photosynthetic model system.

The absorption spectra of chlorophyll b in Triton X-100 micelles at room temperature are superpositions of components with absorption maxima at 640.8, 648.9, 659.5, 669.6, 682.1 and 695.7 nm, obtained from Gaussian analysis of the spectra. The last four forms strongly overlap the chlorophyll a forms of this system obtained with maxima at 659.3, 667.6, 674.3, 680.8, 686.5, 692.8, 701.9, 713.6 and 722.0 nm. Since the in vivo chlorophyll a forms practically coincide with the forms found in this system, the possible existence of in vivo overlapping chlorophyll b and a forms eventually should be taken into consideration. In this case, however, the Gaussian analysis of in vivo absorption bands in itself in the proper spectrum range cannot discriminate between chlorophyll a and b components.     

Spectrophotometric titrations of micellar Schiff''s bases prepared from pyridoxal 5′-phosphate

Electron absorption and equilibrium of the Schiffs bases prepared between pyridoxal 5′-phosphate (PLP) and dodecylamine (DODA) or some other shorter chain amines have been studied in nonionic and cationic micellar solutions with various pH of the bulk solution. In the presence of the nonionic (Triton X-100) micelles the Schiffs bases formed between PLP and DODA were embedded into the micelles because the absorption occured at 335 nm, indicative of the nonpolar milieu. This absorption was constant at pH 5–10. At pH 3–5, the tautomeric form absorbing at 415 nm appeared. This resembles the titration of glycogen phosphorylate or that of Schiffs bases in methanol. Short chain amines absorbed at 415 nm, which is typical of Schiffs bases in aqueous solutions. Tryptophan also absorbed first at 415 nm but the absorption changed to 325 nm with a half-time of ～20 min. This was interpreted as being due to formation of the cyclic structure catalysed by micelles. The pH-dependent equilibrium constant of the reaction between PLP and DODA in Triton X-100 solution had a maximum at pH9, the value being 3500 M^?1, about ten times greater than the value of ethylamine at the same pH. Spectral properties of PLP-DODA imines in the cationic micelles (cetyltrimethylammonium bromide) resembled those in the nonionic micelles, except that at low pH the absorption peak in the 415 nm region did not appear. The equilibrium constant of PLP-DODA had maximum at pH 9, the value being as high as 118000 M^?1. Different properties of nonionic and cationic micelles and the design of micellar model systems of PLP enzymes are discussed.     

New chlorophyll-b forms in a chlorophyll-detergent photosynthetic model system

The absorption spectra of chlorophyll b in Triton X-100 micelles at room temperature are superpositions of components with absorption maxima at 640.8, 648.9, 659.5, 669.6, 682.1 and 695.7 nm, obtained from Gaussian analysis of the spectra. The last four forms strongly overlap the chlorophyll a forms of this system obtained with maxima at 659.3, 667.6, 674.3, 680.8, 686.5, 692.8, 701.9, 713.6 and 722.0 nm.

Since the in vivo chlorophyll a forms practically coincide with the forms found in this system, the possible existence of in vivo overlapping chlorophyll b and a forms eventually should be taken into consideration. In this case, however, the Gaussian analysis of in vivo absorption bands in itself in the proper spectrum range cannot discriminate between chlorophyll a and b components.     

The fluorescence lifetime and quantum yield of chlorophyll a in Triton X-100 solutions

The fluorescence of chlorophyll (Chl) a in 0.007–0.1% Triton X-100 was investigated by a phase-shift technique. The Chl a concentrations varied from 0.7 to 25 μM. Parallel measurements of fluorescence lifetime (τ) and quantum yield (ψ) were made. It was concluded that homogeneous energy transfer takes place at detergent concentrations above 0.025%: (i) the transfer between uniform molecules of the pigment solubilized in Triton X-100 micelles, when τ and ψ are constant; (ii) the transfer towards the quenching centers, resulting in a proportional decrease in τ and ψ. At a Triton X-100 concentration of about 0.025% the Chl a emission becomes heterogeneous. It is evident from the disproportional decrease in τ and ψ (greater in ψ than in τ) and also from the rise of the fluorescence at 730–750 nm. As the Triton X-100 concentration becomes lower than the critical one (0.021%), the number of micelles drops abruptly and Chl a forms colloid particles in the aqueous medium. This manifests itself as a decrease in τ and as a certain stabilization of ψ. Having analyzed the complex pattern of the τ/ψ ratio, we concluded that under these conditions more than 90% of Chl a is in a weakly fluorescent form (τ < 30 ps) and about 1% is in an aggregated state fluorescing at 732 nm with τ about 0.7 ns.     

Studies on mixed micelles of triton X–100 and phosphatidylcholine using nuclear magnetic resonance techniques

The addition of the nonionic detergent Triton X-100 to aqueous phosphatidyl-choline dispersions converts the bilayer structures to mixed micellar structures containing Triton X-100. High-resolution nuclear magnetic resonance spectroscopy at 220 MHz was used to follow this conversion, and the general spectral characteristics of the mixed micelles are presented. The results are discussed in terms of the precise change in structure which occurs as Triton is mixed with the phospholipid bilayers, and it is concluded that, above a molar ratio of about 2:1 Triton to phospholipid, most or all of the phospholipid is in mixed micelles. The relevance of these results to the study of enzymes which require substrate in the form of micelles is discussed.     

The detergent and salt effect on the light-harvesting chlorophyll ab complex from green plants

The light-harvesting accessory pigment-protein complex (LHC) with a chlorophyll (Chl) $\text{a}\text{b}$ ratio of 1.2 was isolated by treating pea chloroplasts with Triton X-100. The LHC was used to investigate the action of ionic (sodium dodecyl sulfate) and non-ionic (Triton X-100) detergents. By optical methods (absorption and fluorescence spectra, measurements of fluorescence yield, ?, and lifetime, τ) two successive stages of the process were demonstrated, namely (1) interaction between detergent monomers and proteins and (2) solubilization of pigments into detergent micelles, which is facilitated by the presence of salts. The concentration ranges, characteristic of these stages, differ by 1.5–2 orders of magnitude for SDS, but slightly overlap for Triton X-100. At the second stage, certain changes occur in LHC absorption and fluorescence spectra. Several stable states of the LHC were established: (1) an aggregated state formed in the presence of 10 mM MgSO₄ with τ ≈ 0.6 ns; (2) the dialyzed LHC with τ ≈ 0.9 ns; (3) the states of the LHC in detergent solution with τ ≈ 2.3, 2.9, 3.4 ns; (4) a 30 kilodalton monomer obtained by SDS-polyacrylamide gel electrophoresis with τ ≈ 4.1 ns. The fluorescence parameters of the LHC states were compared with those of Chl a in detergent micelles (for the micelles τ = 5.6–6.0 ns. The $\text{τ}\text{?}$ ratio (the criterion for emission heterogeneity) for the LHC in the absence of a detergent was shown to be higher at least by a factor of 3.5 than that for Chl a in the presence of a detergent. Successive additions of the detergent to the LHC cause gradual decrease in the $\text{τ}\text{?}$ ratio, and for the LHC monomer it reaches practically the same value as for Chl a in detergent micelles. The results are discussed on the basis of the data obtained previously. It is suggested that in vivo LHCs do not form such aggregates as in water solution without a detergent.     

Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.