Study of different types of chlorophyll a aggregates in solutions and films by absorption and luminescence derivative spectroscopy]

The second derivative of absorption, fluorescence and fluorescence excitation spectra of chlorophyll a in concentrated solutions and films was investigated. More than 14 forms of pigment aggregates, which can be divided into two types--with narrow 8-10nm) and wide (25-40nm) low temperature (-196 degrees C) spectra bands, were found. For the most part of the aggregated forms, the position and half width of the bands, as well as the Stokes shift and relative quantum yield were determined. The comparison of the spectral characteristics points to the indentity of the aggregates and corresponding native forms of Chl. a. It is shown that the universal relationship between absorption and fluorescence bands in applicable to the aggregates of the two types and the energy of resonance interaction between monomers in the aggregates is evaluated.     

Effects of photoproducts on the binding properties of protoporphyrin IX to proteins

Photosensitisers are the photoactive molecules used in photodynamic therapy (PDT) of cancer. Despite the importance of their interaction with polypeptides, only the binding to plasma proteins has been investigated in some detail. In our study we compared the binding of Protoporphyrin IX (a clinically useful photosensitiser) to an immunoglobulin G, with the binding to albumins. Binding to IgG is relevant because a possible method of increasing tumour specificity of photosensitisers is to bind them to tumour-specific antibodies. Binding constants to albumins and the immunoglobulin were comparable ( congruent with6 x 10(-6) M(-1)). The apparent number of PPIX molecules bound to each protein was also within a similar range (from 4 to 7). The absence of a shift in the emission spectrum of PPIX bound to IgG, however, indicates that either larger aggregates of PPIX bind to the immunoglobulin or that the binding site leaves PPIX exposed to the buffer. We observed that PPIX photoproducts compete with PPIX for the same binding sites. The number of PPIX molecules bound to each protein in the presence of photoproducts decreased by 50-80%. Due to the spectral overlap between PPIX and its photoproducts, the binding in the presence of photoproducts was investigated using Derivative Synchronous Fluorescence Spectroscopy (DSFS) to improve the spectral separation between chromophores in solution. We also concluded that fluorescence measurements underestimate the number of PPIX molecules binding each protein. In fact, non-linear Scatchard plots (in the case of albumin binding) by definition yield a minimum number of molecules attached to a protein. Moreover, the binding of large aggregates, formed by an unknown number of PPIX molecules, to IgG results in the underestimate of the number of molecules bound. The number of PPIX molecules bound to these proteins is also much larger than the number of sites estimated by protein fluorescence quenching.     

Spectroscopic detection of adrenaline-quinone formation in micelles

Spectral changes, from 200 nm to 600 nm, of the oxidation of adrenaline to adrenochrome induced by periodate in electrically charged and neutral micelles at pH 3.77 were studied. The observed variations of the peak position, intensity and shape of the fluorescence spectra indicated that depending on the charge of the micelle adrenaline ion is partially embedded into the micellar core. Fluorescence lifetime measurements using Omnilyzer allowed to calculate partition coefficients of 0.36, 0.05 and 0.01 in sodium dodecyl sulphate, tetradodecyltrimethylammonium bromide and Triton X-100, respectively. Kinetics of adrenaline decay during oxidation were followed by its fluorescence what overcame spectral interference in the absorption spectra of adrenaline from the formed intermediates. Scanning absorption spectroscopy, with 100 ms resolution, allowed the recording of spectral changes during the transformation. With this method, the formation of adrenaline-quinone with absorption maxima at 388 nm and 274 nm was detected. The calculated rate constants of the observed kinetics during oxidation were significantly lowered in both charged micelles compared to buffer solution and in Triton X-100 neutral micelles. The observed phenomena are discussed in terms of the electrostatic forces mechanism and in the frame of the Raper-Mason scheme of adrenaline transformation.     

Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes.

The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions.     

Complexation of polymethine dyes with human serum albumin: a spectroscopic study

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Phif; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Phif vary from 10(4) to (5-6) x 10(7) M(-1). K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis-->trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.     

Studies on the temperature effect on bacteriorhodopsin of purple and blue membrane by fluorescence and absorption spectroscopy

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 340 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of bRp.As temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp increases.The binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to bRp.These results suggest that significant structural difference existed between bRband bRp.It was also found that both kinds of bR are highly thermal stable.The maximum wavelength of theprotein fluorescence emission only shifted from 340 nm to 346 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at380 nm formed.The transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high temperature.This phenomenon has not been reported before.     

Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c.

Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.     

Intrinsic fluorescence, difference spectrophotometry and theoretical studies on tertiary structure of calf thymus histone H1

Tyr-72 is included in the hydrophobic cleft which is formed in the histone H1 globular head. Tyr-72 is screened against polar aqueous environment and its intramolecular mobility is sharply retarded. This microenvironment causes a red shift (lambda max = 279 nm) and a sharpening of the longer wavelength shoulder of absorption spectra, a high fluoresence anisotropy value (A = 0,11), high quantum yield of fluoresence (approximately 0.2) and a decrease of the Stern-Volmer Constant during quenching of histone H1 fluorescence by acrylamide. It has been found that the change in the intensity of histone fluorescence at lambda excit = 265 nm, but not at lambda excit = 280 nm, is due to the changes in the quantum yield of fluorescence. The increase of fluorescence intensity at lambda excit = 280 nm depends on the changes in the quantum yield and molar extinction coefficient of histone H1 tyrosyl chromophore. The change in the ratio of fluorescence intensity exited at 280 nm (F280) to the fluorescence intensity excited at 265 nm (F265) corresponds to the change of delta epsilon 286 in difference absorption spectra. The introduction of the parameter Cf = F280/F265 allows one to go over to studying excitation spectrum shifts instead of histone absorption spectrum shifts, which is much more convenient methodologically since in this case it is possible to carry out research using lower protein concentrations and turbid solutions. The results make it possible to designate Tyr-72 of histone H1 as a special class of fluorescent tyrosyls whose properties differ from those of tyrosyls of other tryptophane-free proteins: RNAase, insulin, core histones--H2A, H2B, H3, H4 and some others.     

A structural study of bovine lens alpha-crystallin and its subunits by absorption and linear dichroism spectroscopy

The structure of the bovine alpha-crystallin aggregate and its reaggregated isolated subunits has been studied by measurement of their absorption and linear dichroism spectra over the range 250-350 nm. Also, changes in structure with respect to time have been monitored in this way. From the absorption spectra it appears that the aromatic residues in subunit aggregates are in the same chemical environment as those in native protein. The light scattering due to the size of the protein molecules increases when the proteins are kept in solution, this effect being much stronger for the subunits. The linear dichroism spectra point to strong structural ordering in alpha-crystallin, the absorption transition dipoles of the aromatic residues being preferentially aligned along the long axis of the molecules. Moreover, a considerable deviation from a spherical or tetrahedrally symmetric structure of alpha-crystallin is inferred. The subunit aggregates show less ordering and might be more spherical. When kept in solution, their structural order seems to be decreased. The linear dichroism spectra show absorption at 325 nm, which is not detectable in the normal absorption spectra.     

Protoporphyrin ix as a possible ancient photosensitizer: Spectral and photochemical studies

Due to the potential special position of protoporphyrin IX in the evolution of photosynthesis, the absorption and fluorescence characteristics of this pigment and its complexes with human serum albumin (HSA) and basic proteinoid have been studied in parallel with their photochemical activity. The most significant change in the absorption spectrum of PP IX was the appearance of a new maximum at 455 (or 461) nm in the presence of HSA or proteinoid respectively. Some changes in the physicochemical properties of PP IX in different microenvironments have been detected by changes in fluorescence emission and excitation spectra (intensity, quantum yields, position of maxima). The increase of fluorescence quantum yield resulting from the formation of PP IX complexes with HSA or proteinoid correlates with the increase of their photochemical activity. Results obtained are discussed from the point of view of the early evolution of the photosynthetic apparatus.     

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.     

Ligand binding at membrane mimetic interfaces. Human serum albumin in reverse micelles.

The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2-ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x 10(4) M-1 cm-1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Ka approximately 1.0 x 10(5) M-1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant. These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine-411 does not seem substantially modified by the 15% decrease affecting the alpha helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels.     

Protochlorophyll forms with different molecular arrangements

Spectral properties of protochlorophyll (PChl) forms were investigated in solid-film model systems by absorption. fluorescence and circular dichroism (CD) spectroscopy. The solid films were prepared from diethyl ether solution of PChl on a cover glass surface by evaporation of the solvent. After preparation the films usually showed an absorption maximum at 635 nm or in some cases at 640 nm. The PChl form with 635 nm absorption maximum had no CD signal, whilst the films with absorption maximum at 640 nm gave an intense negative CD band at about 640 nm and a positive one at 668 nm. The treatment of the films with ammonia or acetone vapour resulted in a red shift of the absorption maximum from 635 nm or 640 nm to 650 nm. The study of the CD spectra of the films with different PChl forms showed that, depending on the treatment, forms of PChl with similar absorption and fluorescence spectra, but with opposite CD signals, can exist. It is suggested that the differences of the CD spectra are mainly due to different arrangements of the aggregates.     

pH-dependent changes in the tryptophan and porphyrin fluorescence of the apomyoglobin complex with protoporphyrin IX and methemoglobin

The fluorescence of protoporphyrin IX (PPIX) complexed with sperm whale apomyoglobin as well as the tryptophan fluorescence of this complex and of metmyoglobin within the pH range of 3.5-13 was studied. It was shown that an increase in pH from 5.3 to 10.8 does not influence the fluorescence of PPIX in the complex and causes no essential changes in the fluorescence of Trp residues, which occur at more acidic and, correspondingly, alkaline pH values simultaneously with the protein denaturation. This is accompanied by a sharp increase in the quantum yield of tryptophan fluorescence due to dissociation of PPIX from the complex. Similar changes are observed in metMb at pH less than 4.3 and greater than 12 which is concomitant with absorption changes in the Soret band, thus indicating a higher stability of metMb towards the acid and alkaline denaturation as compared to the complex. In both cases, a slight alteration in the shape of the tryptophan fluorescence spectrum is observed, which precedes alkaline denaturation of the Mb molecule and is probably due to changes in the conformation of the N-terminal region caused by the break of the salt bridges stabilizing the native structure of the protein.     

Emission spectra from copper proteins containing type-3 centres

Aqueous solutions of copper-proteins containing type-3 centres (ceruloplasmin, tyrosinase, haemocyanin), excited within their absorption bands at 325-345 nm, show typical luminescence spectra. The emission bands peak at 415-445 nm and their decay time is no longer than 10 ns. A strong analogous fluorescence is obtained also by excitation of concentrated solutions of carboxylic acids and amino acids, which show again absorption bands around 330 nm. Such a fluorescence, although less intense, is also observed in copper(II) carboxylate solutions. In contrast, no fluorescence has been recorded in solutions of acetic anhydride and of polypeptides (valinomycin, gramicidin D), which do not have free carboxyl groups. We tentatively attribute this novel fluorescence in the investigated copper proteins to interactions between carboxyl groups of amino acids at, or near, the active site.     

Influence of Silver Colloid Presence on Stilbazolium Merocyanine Emission

Absorption, fluorescence emission, and fluorescence excitation spectra of stilbazolium merocyanine (1-(n-butyl)-4[(3,5-dimethoxy-4-oxocyclohexa-2,5-dienylidene)ethylidene]-1,4-dihydropyridyne) dye in water solution without and with colloidal silver addition were measured. In the presence of the colloid, besides the absorption band assigned to the protonated species of the dye (at 391 nm), an absorption band related to the free-base species appears at 490 nm. From the absorption and emission spectra, measured at various dye concentrations, follows that the aggregates are not effectively formed. Therefore, the long-wavelength absorption and fluorescence bands have to be related to some dye forms created by the solvatochromic effects. The fluorescence bands of the protonated and the free-base species are located at 559 nm and at about 630 nm, respectively. The shape of the long-wavelength band suggests the occurrence of more than one free-base form of the dye. At some dye and colloid concentrations, the global emission of the sample is enhanced as a result of silver addition. The increase in the emission yield of the dye could be partially due to not only the change in the concentrations of dye forms exhibiting various emission yields but is also due to the resonance surface plasmon effect. Because of the superposition of several effects, before the practical application of merocyanine as an indicator of metal presence in biological samples, its spectral properties in the system investigated should be established.     

Oligomers of bacteriochlorophyll and bacteriopheophytin with spectroscopic properties resembling those found in photosynthetic bacteria

Oligomers of bacteriopheophytin (BPh) and bacteriochlorophyll (BChl) were formed in mixed aqueous-organic solvent systems, and in aqueous micelles of the detergent lauryldimethylamine oxide (LDAO). Conditions were found that gave relatively homogeneous samples of oligomers and that allowed quantitative comparisons of the spectroscopic properties of the monomeric and oligomeric pigments. The formation of certain types of oligomers is accompanied by a large bathochromic shift of the long-wavelength (Q_y) absorption band of the BChl or BPh, and by a substantial increase in its dipole strength (hyperchromism). The hyperchromism of the Q_y band occurs at the expense of the Soret band, which loses dipole strength. The Q_x band shifts slightly to shorter wavelengths and also loses dipole strength. The CD spectrum in the near-infra-red (Q_y) region becomes markedly nonconservative. (The net rotational strength in the Q_y region is positive.) This also occurs at the expense of the bands at shorter wavelengths, which gain a net negative rotational strength. The spectroscopic properties of the oligomers resemble those of some of the BChl-protein complexes found in photosynthetic bacteria. The oligomerization of BPh in LDAO micelles is linked to the formation of large, cylindrical micelles that contain on the order of 10⁵ LDAO molecules. However, the spectral changes probably occur on the formation of small oligomers of BPh; they begin to be seen when the micelles contain about 10 molecules of BPh. The BPh oligomers formed in LDAO micelles fluoresce at 865 nm, but the fluorescence yield is decreased about 40-fold, relative to that of monomeric BPh. The fluorescence yield is insensitive to the BPh/LDAO molar ratio, suggesting that the oligomers formed under these conditions are predominantly dimers. When the oligomers are excited with a short flash of light, they are converted with a low quantum yield into a metastable form. This transformation probably involves alterations in the geometry of the oligomer, but not full dissociation.     

Origin of laurdan sensitivity to the vesicle-to-micelle transition of phospholipid-octylglucoside system: a time-resolved fluorescence study

The fluorescent probe laurdan has been shown to be sensitive to the vesicle-to-micelle transition of phosphatidylcholine/octylglucoside (M. Paternostre, O. Meyer, C. Grabielle-Madelmont, S. Lesieur, and, Biophys. J. 69:2476-2488). On the other hand, a study on the photophysics of laurdan in organic solvents has shown that the complex de-excitation pathway of the probe can be described by two successive processes, i.e., an intramolecular charge transfer followed by dielectric relaxation of the solvent if polar. These two excited-state reactions lead to three emitting states, i.e., a locally excited state, a charge transfer state, and a solvent relaxed state (M. Viard, J. Gallay, M. Vincent, B. Robert and, Biophys. J. 73:2221-2234). Experiments have been performed using time-resolved fluorescence on the probe inserted in amphiphile aggregates (mixed liposomes, mixed micelles) different in detergent-to-lipid ratios. The results have been compared with those obtained for laurdan inserted in dipalmitoyl phosphatidylcholine liposomes in the gel and in the fluid lamellar phase. Except for laurdan in dipalmitoyl phosphatidylcholine liposomes in the gel lamellar phase, the red part of the emission spectra originates from the de-excitation of the relaxed excited state of laurdan, indicating that indeed the dielectric relaxation process is an important phenomena in the ground-state return pathway of this probe. On the other hand, the maximization entropy method (MEM) analysis of the fluorescence decay recorded in the blue part of the emission spectra indicates that the dielectric relaxation is not the only reaction occurring to the excited state of laurdan. Moreover, the analysis of the fluorescence decays of laurdan inserted in gel lamellar dipalmitoylphosphatidylcholine (DPPC) liposomes indicates excited-state reactions, although dielectric relaxation is impossible. These results are in agreement with the de-excitation pathway determined from laurdan behavior in organic solvent even if, in most of the aggregates studied in this work, the major phenomenon is the dielectric relaxation of the solvent. All along the vesicle-to-micelle transition, we have observed that the lifetime of the relaxed excited state of laurdan continuously decreases probably due to a dynamic quenching process by water molecules. On the other hand, the time constant of the dielectric relaxation process remains almost unchanged in the lamellar part of the transition but abruptly decreases as soon as the first mixed micelle is formed. This decrease is continuous all over the rest of the transition even if it is more pronounced in the mixed liposomes' and mixed micelles' coexistence. The increase of the octylglucoside-to-lipid ratio of the mixed micelles via the change of the size and the shape of the aggregates may facilitate the penetration and the mobility of water molecules. Therefore, during the vesicle-to-micelle transition, laurdan probes the evolution of both the amphiphile packing in the aggregates and the increase of the interface polarity. This study finally shows that the detergent-to-lipid ratio of the mixed micelles is an important parameter to control to limit the penetration and the mobility of water within the amphiphile aggregates and that laurdan is a nice tool to monitor this phenomenon.     

Spectral properties and inclusion of a hetero-chalcone analogue in organized media of micellar solutions and beta-cyclodextrin

The absorption and fluorescence emission spectral properties of 3-(4'-dimethylaminophenyl)-1-(2-thienyl)prop-2-en-1-one, abbreviated as DMATP, have been investigated in organized media of aqueous micellar and beta-cyclodextrin (CD) solutions. While the absorption spectra are less sensitive to the nature of the added surfactant or CD, the characteristics of the intramolecular charge transfer (ICT) fluorescence are highly sensitive to the properties of the medium. The ICT maximum is strongly blue-shifted with a great enhancement in the fluorescence quantum yield on adding micellar or CD solutions. This indicates the solubilization of DMATP in the micellar core and formation of an inclusion complex with beta-CD. The critical micelle concentration (CMC) as well as the polarity of the micellar core of SDS, CTAB and TX-100 have been determined. The CMC values are in good agreement with the reported values while the polarity is lower indicating that DMATP molecules are incorporated in the micellar core not at the micellar interface. The inclusion constants of binding of DMATP in micellar or CD have been also determined. The thermodynamic parameters of formation of DMATP:CD inclusion complex have been calculated from the temperature dependence of the fluorescence spectra of the formed complex. The negative enthalpy and free energy of formation indicate that the inclusion process is energetically favorable. The highly negative value of formation entropy (DeltaS = -162.3 J mol(-1) K(-1)) reflects the high restrictions imposed on the movement of both the host and included guest molecules which is consistent with the increase of the fluorescence yield and blue shift of the fluorescence maximum.     

DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.

Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.