Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet–vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (C_BZC/C_Lys < 9) and a combined quenching process at higher concentration of BZC (C_BZC/C_Lys > 9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn²⁺ on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied.     

Tunable Molecular Logic Gates Designed for Imaging Released Neurotransmitters

Tunable dual‐analyte fluorescent molecular logic gates (ExoSensors) were designed for the purpose of imaging select vesicular primary‐amine neurotransmitters that are released from secretory vesicles upon exocytosis. ExoSensors are based on the coumarin‐3‐aldehyde scaffold and rely on both neurotransmitter binding and the change in environmental pH associated with exocytosis to afford a unique turn‐on fluorescence output. A pH‐functionality was directly integrated into the fluorophore π‐system of the scaffold, thereby allowing for an enhanced fluorescence output upon the release of labeled neurotransmitters. By altering the pH‐sensitive unit with various electron‐donating and ‐withdrawing sulfonamide substituents, we identified a correlation between the pK_a of the pH‐sensitive group and the fluorescence output from the activated fluorophore. In doing so, we achieved a twelvefold fluorescence enhancement upon evaluating the ExoSensors under conditions that mimic exocytosis. ExoSensors are aptly suited to serve as molecular imaging tools that allow for the direct visualization of only the neurotransmitters that are released from secretory vesicles upon exocytosis.     

FRET‐Based Mitochondria‐Targetable Dual‐Excitation Ratiometric Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells

Hydrogen sulfide (H₂S) is connected with various physiological and pathological functions. However, understanding the important functions of H₂S remains challenging, in part because of the lack of tools for detecting endogenous H₂S. Herein, compounds Ratio‐H₂S 1/2 are the first FRET‐based mitochondrial‐targetable dual‐excitation ratiometric fluorescent probes for H₂S on the basis of H₂S‐promoted thiolysis of dinitrophenyl ether. With the enhancement of H₂S concentration, the excitation peak at λ≈402 nm of the phenolate form of the hydroxycoumarin unit drastically increases, whereas the excitation band centered at λ≈570 nm from rhodamine stays constant and can serve as a reference signal. Thus, the ratios of fluorescence intensities at λ=402 and 570 nm (I₄₀₂/I₅₇₀) exhibit a drastic change from 0.048 in the absence of H₂S to 0.36 in the presence of 180 μM H₂S; this is a 7.5‐fold variation in the excitation ratios. The favorable properties of the probe include the donor and acceptor excitation bands, which exhibit large excitation separations (up to 168 nm separation) and comparable excitation intensities, high sensitivity and selectivity, and function well at physiological pH. In addition, it is demonstrated that the probe can localize in the mitochondria and determine H₂S in living cells. It is expected that this strategy will lead to the development of a wide range of mitochondria‐targetable dual‐excitation ratiometric probes for other analytes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.     

Monitoring intracellular pH fluctuation with an excited-state intramolecular proton transfer-based ratiometric fluorescent sensor

Intracellular pH is a key parameter related to various biological and pathological processes. In this study, a ratiometric pH fluorescent sensor ABTT was developed harnessing the amino-type excited-state intramolecular proton transfer (ESIPT) process. Relying on whether the ESIPT proceeds normally or not, ABTT exhibited the yellow fluorescence in acidic media, or cyan fluorescence in basic condition. According to the variation, ABTT behaved as a promising sensor which possessed fast and reversible response to pH change without interference from the biological substances, and exported a steady ratiometric signal (I₄₇₈/I₅₄₆). Moreover, due to the ESIPT effect, large Stokes shift and high quantum yield were also exhibited in ABTT. Furthermore, ABTT was applied for monitoring the pH changes in living cells and visualizing the pH fluctuations under oxidative stress successfully. These results elucidated great potential of ABTT in understanding pH-dependent physiological and pathological processes.     

A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria

The measurement of physicochemical parameters in living cells can provide information on individual cellular organelles, helping us to understand subcellular function in health and disease. While organelle‐specific chemical probes have allowed qualitative evaluation of microenvironmental variations, the simultaneous quantification of mitochondrial local microviscosity (η_m) and micropolarity (?_m), along with concurrent structural variations, has remained an unmet need. Herein, we describe a new multifunctional mitochondrial probe ( MMP ) for simultaneous monitoring of η_m and ?_m by fluorescence lifetime and emission intensity recordings, respectively. The MMP enables highly precise measurements of η_m and ?_m in the presence of a variety of agents perturbing cellular function, and the observed changes can also be correlated with alterations in mitochondrial network morphology and motility. This strategy represents a promising tool for the analysis of subtle changes in organellar structure.     

A Novel Fluorescent Probe for Hydrogen Peroxide and Its Application in Bio-Imaging

Hydrogen peroxide (H₂O₂) plays an important role in the human body and monitoring its level is meaningful due to the relationship between its level and diseases. A fluorescent sensor (CMB) based on coumarin was designed and its ability for detecting hydrogen peroxide by fluorescence signals was also studied. The CMB showed an approximate 25-fold fluorescence enhancement after adding H₂O₂ due to the interaction between the CMB and H₂O₂ and had the potential for detecting physiological H₂O₂. It also showed good biocompatibility and permeability, allowing it to penetrate cell membranes and zebrafish tissues, thus it can perform fluorescence imaging of H₂O₂ in living cells and zebrafish. This probe is a promising tool for monitoring the level of H₂O₂ in related physiological and pathological research.     

A Reversible Fluorescent Probe for Real-Time Quantitative Monitoring of Cellular Glutathione

The ability to monitor and quantify glutathione (GSH) in live cells is essential in order to gain a detailed understanding of GSH-related pathological events. However, owing to their irreversible response mechanisms, most existing fluorescent GSH probes are not suitable for this purpose. We have developed a ratiometric fluorescent probe (QG- 1 ) for quantitatively monitoring cellular GSH. The probe responds specifically and reversibility to GSH with an ideal dissociation constant (K_d) of 2.59 mm and a fast response time (t_1/2=5.82 s). We also demonstrate that QG- 1 detection of GSH is feasible in a model protein system. QG- 1 was found to have extremely low cytotoxicity and was applied to determine the GSH concentration in live HeLa cells (5.40±0.87 mm ).     

Exploring Reversible Quenching of Fluorescence from a Pyrazolo[3,4‐b]quinoline Derivative by Protonation

Pyrazolo[3,4‐b]quinoline derivatives are reported to be highly efficient organic fluorescent materials suitable for applications in light‐emitting devices. Although their fluorescence remains stable in organic solvents or in aqueous solution even in the presence of H₂O, halide salts (LiCl), alkali (NaOH) and weak acid (acetic acid), it suffers an efficient quenching process in the presence of protic acid (HCl) in aqueous or ethanolic solution. This quenching process is accompanied by a change in the UV spectrum, but it is reversible and can be fully recovered. Both steady‐state and transient fluorescence spectra of 1‐phenyl‐3,4‐dimethyl‐1H‐pyrazolo‐[3,4‐b]quinoline (PAQ5) during quenching are measured and analyzed. It is found that a combined dynamic and static quenching mechanism is responsible for the quenching processes. The ground‐state proton‐transfer complex [PAQ5 ??? H⁺] is responsible for static quenching. It changes linearly with proton concentration [H⁺] with a bimolecular association constant K_S=1.95 M ^?1 controlled by the equilibrium dissociation of HCl in ethanol. A dynamic quenching constant K_D=22.4 M ^?1 is obtained by fitting to the Stern–Volmer equation, with a bimolecular dynamic quenching rate constant k_d=1.03×10⁹ s^?1 M ^?1 under ambient conditions. A change in electron distribution is simulated and explains the experiment results.     

Accelerated Regeneration of ATP Level after Irradiation in Human Skin Fibroblasts by Coenzyme Q10

Human skin is exposed to a number of harmful agents of which the ultraviolet (UV) component of solar radiation is most important. UV‐induced damages include direct DNA lesions as well as oxidative damage in DNA, proteins and lipids caused by reactive oxygen species (ROS). Being the main site of ROS generation in the cell, mitochondria are particularly affected by photostress. The resulting mitochondrial dysfunction may have negative effects on many essential cellular processes. To counteract these effects, coenzyme Q₁₀ (CoQ₁₀) is used as a potent therapeutic in a number of diseases. We analyzed the mitochondrial respiration profile, the mitochondrial membrane potential and cellular ATP level in skin fibroblasts after irradiation. We observed an accelerated regeneration of cellular ATP level, a decrease in mitochondrial dysfunction as well as a preservation of the mitochondrial membrane potential after irradiation in human skin fibroblasts by treatment with CoQ₁₀. We conclude that the faster regeneration of the ATP level was achieved by a preservation of mitochondrial function by the addition of CoQ₁₀ and that the protective effect of CoQ₁₀ is primarily mediated via its antioxidative function. We suggest also that it might be further dependent on a stimulation of DNA repair enzymes by CoQ₁₀.     

Partitioning of unsaturated hydrophilic monomer in microemulsion media monitored by pyrene fluorescence method

The extent of intra‐ and interchain associations of (un)charged water‐soluble monomers in the homogeneous and micellar solutions was studied with steady‐state fluorescence spectroscopy. Fluorescence spectroscopic experiments were performed on uncharged (acryl amide) and charged hydrophilic monomers [zwitterionic 3‐dimethyl(methacryloyloxyethyl)ammonium propane sulfonate (DMAPS), etc.] with pyrene as a probe. In both the homogeneous and micellar solutions, linear Stern–Volmer plots were obtained that implied that the quenching process can be considered as totally dynamic. The Stern–Volmer constant (K_SV) for DMAPS decreased with an increasing dielectric constant of solvent and the concentration of simple electrolyte. An abrupt decrease in K_SV was observed in the presence of a small amount of anionic emulsifier [below the critical micelle concentration (cmc)]. The dependence of K_SV on pH for DMAPS was described by a curve with a maximum at about pH = 7. This was interpreted in terms of segregation of DMAPS and the variation of a optimal microenvironment for the probe and quencher with pH. The quenching rate in the micellar solutions strongly increased above the cmc but was lower than that in the homogeneous solutions. In the micellar solutions (above the cmc), the microenvironment for an interaction between the probe and quencher was suggested to be the whole microdroplet. The dependence of K_SV on pH for DMAPS is described by a curve with a maximum at about pH = 9.3. The synergistic effect arises from the segregation of charged quencher molecules within the microdroplets. The complex (or strong interaction) between quencher and additive(s) is supposed to increase the dynamic nature of microdroplets that provides an optimal microenvironment for probe and quencher. A good coemulsifier, however, removes quencher from the interface and creates a barrier for entering monomer (quencher) into the core of micelles; therefore, quenching is depressed. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 571–581, 2003     

The deexcitation of the S1 state of aminoanthraquinones: A steadystate and timeresolved study

The non-radiative processes of deactivation from the lowest singlet excited state of aminoanthraquinones have been studied using steady-state and time-resolved methods. The fluorescence decay rate constant, k_f correlates well with the solvent polarity parameter, E_T(30), in nonhydrogen bonding solvents. Large deuterium isotope effects in fluorescence lifetimes (τ_f) and quantum yields (ϕ_f) are observed in the case of 1-amino (AAQ) and 1-methylaminoanthraquinones (MAQ), where the S₁ state is mainly deactivated through internal conversion to the ground state. The temperature-dependence of the fluorescence quantum yields of various aminoanthraquinones was also investigated. The ϕ_f and τ_f exhibited strong temperature-dependence in the case of 1-acetylaminoanthraquinone (ACAQ). In the case of ACAQ, the intersystem crossing to the triplet state is a major deactivation channel from the S₁ and in this derivative a close-lying T₂ state seems to be responsible for the high k_isc rate. The fluorescence properties of 1,5-diaminoanthraquinone (DAQ) are affected by intermolecular hydrogen bonding with alcohols. Increasingn-alkyl chain length in the case of l-(n-alkyl)aminoanthraquinones from methyl to butyl does not produce any change in the fluorescence properties, whereas a hydroxypropyl substitution results in a small decrease of ϕ_f and τ_f in these compounds, indicating an interaction of the hydroxyl group with the carbonyl group of the aminoanthraquinones.     

Effects of TiO<Subscript>2</Subscript> surface fluorination on photocatalytic degradation of methylene blue and humic acid

Photocatalytic degradation (PCD) reactions of cationic methylene blue (MB) and anionic humic acid (HA) were studied in naked TiO₂ and fluorinated TiO₂ (F-TiO₂) suspensions in order to investigate how the modification of the TiO₂ surface functional group influenced PCD reactions. Adsorption behaviors of MB and HA in the naked TiO₂ followed a typical pH-dependent electrostatic interaction mechanism. On the other hand, those in the F-TiO₂ were markedly changed and even showed a reversed dependence in specific pH ranges due to surface fluoride interrupting the interaction of substrates and surface titanol groups. PCD rates of MB (k _MB) and its N-demethylation (Δλ _max) were significantly increased by surface fluorination below circum-neutral pH range, in particular, by a factor of 12 and 54 at pH 2, respectively. In the case of HA, the fluorination had an insignificant effect on its degradation rate but appeared to change its degradation behavior. It has been suggested that, although the primary effect of fluorination enhances the photocatalytic production of hydroxyl radicals, the change in electrostatic interaction with substrates could affect PCD as well.     

Water‐Soluble Red‐Emitting Distyryl‐Borondipyrromethene (BODIPY) Dyes for Biolabeling

A series of water‐soluble red‐emitting distyryl‐borondipyrromethene (BODIPY) dyes were designed and synthesized by using three complementary approaches aimed at introducing water‐solubilizing groups on opposite faces of the fluorescent core to reduce or completely suppress self‐aggregation. An additional carboxylic acid functional group was introduced at the pseudo‐meso position of the BODIPY scaffold for conjugation to amine‐containing biomolecules/biopolymers. The optical properties of these dyes were evaluated under simulated physiological conditions (i.e., phosphate‐buffered saline (PBS), pH 7.5) or in pure water. The emission wavelength (λ_max) of these labels was found in the 640–660 nm range with quantum yields from modest to unprecedentedly high values (4 to 38 %). The bioconjugation of these distyryl‐BODIPY dyes with bovine serum albumin (BSA) and the monoclonal antibody (mAb) 12A5 was successfully performed under mild aqueous conditions.     

Synthesis and Characterization of a Water‐Soluble Carboxylated Polyfluorene and Its Fluorescence Quenching by Cationic Quenchers and Proteins

We have developed a new intermediate monomer, 2,7‐[bis(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)‐9,9‐bis(3‐(tert‐butyl propanoate))]fluorene, that allows the easy synthesis of water‐soluble carboxylated polyfluorenes. As an example, poly[9,9′‐bis(3′′‐propanoate)fluoren‐2,7‐yl] sodium salt was synthesized by the Suzuki coupling reaction, and the properties of the polymer were studied in aqueous solutions of different pH. Fluorescence quenching of the polymer by different cationic quenchers (MV²⁺, MV⁴⁺, and NO₂MV²⁺; MV=methyl viologen) was studied, and the quenching constants were found to be dependent on the charge and electron affinity of the quencher molecule and the pH of the medium. The largest quenching constant was observed to be 1.39×10⁸ M ^?1 for NO₂MV²⁺ at pH 7. The change in polymer fluorescence upon interaction with different proteins was also studied. Strong fluorescence quenching of the polymer was observed in the presence of cytochrome c, whereas weak quenching was observed in the presence of myoglobin and bovine serum albumin. Lysozyme quenched the polymer emission at low protein concentrations, and the quenching became saturated at high protein concentrations. Under similar experimental conditions, the polymer showed improved quenching efficiencies toward cationic quenchers and a more selective response to proteins relative to other carboxylated conjugated polymers.     

Fluorescence Quenching Study on the Interaction of Some Schiff Base Complexes with Bovine Serum Albumin

The interaction of Schiff base ligand A and its three metal complexes [A‐Fe(II), A‐Cu(II), and A‐Zn(II)] with bovine serum albumin (BSA) was investigated using a tryptophan fluorescence quenching method. The Schiff base ligand A and its three metal complexes all showed quenching of BSA fluorescence in a Tris‐HCl buffer. Quenching constants were determined for quenching BSA by the Schiff base ligand A and its metal complexes in a Tris‐HCl buffer (pH=7.4) at different temperatures. The experimental results show that the dynamic quenching constant (K_SV) was increased with increasing temperature, whereas the association constant (K) was decreased with the increase of temperature. The thermodynamic parameters ΔH, ΔG and ΔS at different temperatures were calculated. The ionic strength of the Tris‐HCl buffer had a great influence on the wavelength of maximum emission of BSA. Under low ionic strength, the emission spectra of BSA influenced by A‐Zn(II) had a small blue shift. Compared to A‐Zn(II), the emission spectra of BSA in the presence of the Schiff base ligand A and A‐Cu(II) had no significant λ_em shift. At high ionic strength, the emission spectra of BSA upon addition of the Schiff base A, A‐Fe(II), and A‐Zn(II) all had a red shift, but the emission spectra of BSA had λ_em shift neither at low ionic strength, nor at high ionic strength in the presence of A‐Cu(II). Furthermore, the temperature did not affect the λ_em shift of BSA emission spectra.     

New environmentally responsive fluorescent N‐isopropylacrylamide copolymer and its application to DNA sensing

We report two novel multifunctional copolymers consisting of a temperature‐responsive poly(N‐isopropylacrylamide) (PNIPAA) segment and a fluorescent fluorene‐containing acrylic polymer segment with pH responsiveness and/or DNA‐sensing ability. The functional acrylic monomer with a fluorene dimer side group substituted with amino units was synthesized first. Then, it was copolymerized with N‐isopropylacrylamide to result in a new water‐soluble, fluorescent PNIPAA copolymer ( P1 ). The fluorescent properties of P1 under neutral and acidic conditions did not change with the temperature. However, significant variation was observed under basic conditions. The protonation of the amino moiety at a low pH improved the solubility and prevented aggregation for fluorescence quenching, but not under the basic conditions. Although aggregation of the fluorene units was significant at room temperature under basic conditions, the aggregation was resolved at a temperature above the lower critical solution temperature. These findings indicated the pH‐ and temperature‐responsive characteristics of P1 . Moreover, after the amino groups were quaternized, the obtained polymer could be used as a biosensor because the fluorescence intensity was quenched with the addition of DNA. This study demonstrates that multifunctional materials with pH‐ and temperature‐sensing characteristics and biological molecules could be realized by the incorporation of a functional fluorene‐containing moiety with PNIPAA. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5495–5504, 2006     

Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons

An enzyme-based biosensor was developed by co-immobilization of purified enzyme haloalkane dehalogenase (EC 3.8.1.5) and a fluorescence pH indicator on the tip of an optical fiber. Haloalkane dehalogenase catalyzes hydrolytic dehalogenation of halogenated aliphatic hydrocarbons, which is accompanied by a pH change influencing the fluorescence of the indicator. The pH sensitivity of several fluorescent dyes was evaluated. The selected indicator 5(6)-carboxyfluorescein was conjugated with bovine serum albumin and its reaction was tested under different immobilization conditions. The biosensor was prepared by cross-linking of the conjugate in tandem with haloalkane dehalogenase using glutaraldehyde vapor. The biosensor, stored for 24 h in 50 mM phosphate buffer (pH 7.5) prior to measurement, was used after 15 min of equilibration, the halogenated compound was added, and the response was monitored for 30 min. Calibration of the biosensor with 1,2-dibromoethane and 3-chloro-2-(chloromethyl)-1-propene showed an excellent linear dependence, with detection limits of 0.133 and 0.014 mM, respectively. This biosensor provides a new tool for continuous in situ monitoring of halogenated environmental pollutants.     

Angelica gigas NAKAI and Its Active Compound,Decursin, Inhibit Cellular Injury as an Antioxidant by the Regulation of AMP-Activated Protein Kinase and YAP Signaling

Natural products and medicinal herbs have been used to treat various human diseases by regulating cellular functions and metabolic pathways. Angelica gigas NAKAI (AG) helps regulate pathological processes in some medical fields, including gastroenterology, gynecology, and neuropsychiatry. Although some papers have reported its diverse indications, the effects of AG against arachidonic acid (AA)+ iron and carbon tetrachloride (CCl₄) have not been reported. In HepG2 cells, AA+ iron induced cellular apoptosis and mitochondrial damage, as assessed by mitochondrial membrane permeability (MMP) and the expression of apoptosis-related proteins. On the other hand, AG markedly inhibited these detrimental phenomena and reactive oxygen species (ROS) production induced by AA+ iron. AG activated the liver kinase B1 (LKB1)-dependent AMP-activated protein kinase (AMPK), which affected oxidative stress in the cells. Moreover, AG also regulated the expression of yes-associated protein (YAP) signaling as mediated by the AMPK pathways. In mice, an oral treatment of AG protected against liver toxicity induced by CCl₄, as indicated by the plasma and histochemical parameters. Among the compounds in AG, decursin had antioxidant activity and affected the AMPK pathway. In conclusion, AG has antioxidant effects in vivo and in vitro, indicating that natural products such as AG could be potential candidate for the nutraceuticals to treat various disorders by regulating mitochondrial dysfunction and cellular metabolic pathways.     

Photophysical and rheological properties of naphthalene-labeled cationic poly(dimethyl sulfate quaternized acrylamide/N,N-dimethylaminopropylmaleimide copolymer)

The synthesis, rheological, and fluorescence properties of a cationic water-soluble copolymer, naphthalene-labeled cationic poly(dimethyl sulfate quaternized acrylamide/N,N-dimethylaminopropylmaleimide copolymer), poly(DSQADMAPM)/NA, are reported. When fluorescent hydrophobes (naphthyl group) are incorporated into the cationic copolymer, the photophysical response may effectively probe solution behavior on the microscopic level. The salt and pH responsiveness inherent to the cationic copolymer systems is a function of ionic group type. Experimental results indicate that I_E/I_M increases steadily with increases in polymer concentration and I_E/I_M values for a given polymer concentration are higher in salt. At low pH values, I_E/I_M is high and excimer emission increases as the quaternary amino groups (R₄N⁺) are screened out. Dynamic light scattering (QELS) measurements indicate that diffusion coefficients of the cationic copolymer increase and the hydrodynamic diameters decrease with increasing salt concentration. Viscosity studies reveal that the polymer coil shrinks as salt is added. In fluorescence quenching study, the reduction in the quenching efficiency of thallium (Tl⁺) with salt addition can arise from enhanced compartmentalization of naphthalene labels as added electrolyte enhances intrapolymer micellization. The intrapolymer micelle is easily formed, indicating that the thallium ion has difficulty in reacting with bound naphthalenes located in the shrunk polymer coil. The cationic copolymer is depicted as an expanded polymer coil in deionized water because of intra- and interchain repulsions. Consequently, salt addition breaks down the repulsions and enhances intrapolymer micellization. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36 : 11–19, 1998     

The Effects of pH and Ionic Strength on the Aggregation of Bacteriochlorophyll c in Aqueous Organic Media: The Possibility of Two Kinds of Aggregates

The aggregation behavior of two homologs of bacteriochlorophyll c (BChl c) in various media was investigated for the effects of pH and salt, and the corresponding structures were analyzed by Fourier transform (FT)-IR spectroscopy. R-[P, E] BChl c_F (3¹-R-form of BChl c with a propyl group at the C-8 position and an ethyl group at the C-12 position) and R-[E, E] BChl c_F (3¹-R-form of BChl c with two ethyl groups at positions C-8 and C-12) were isolated from the green sulfur bacterium Chloro-bium limicola. Aggregates of each homolog showed a pH-dependent shift of the absorption maximum; at low pH, the peak moved to the red. This tendency was also revealed by circular dichroic spectra. A similar red shift of the peak was also induced by a high concentration of salt (NaCl) or buffer for both homologs. The FT-IR spectrum indicates that at low pH, both homologs formed a rather amorphous aggregate. On the other hand, a regular structure of R-[P, E] BChl c_F was indicated in an acetone-water mixture. This structure was stabilized by a triangular interaction among three pigment molecules through the Mg-OH (3>) O = C (13¹) linkage. This structure was not found for R-[E, E] BChl c_F. These results indicate that the replacement of the side chain at the C-8 position on the macrocycle induces a change in aggregation behavior. A possible heterogeneity of the in vivo rod structure of chlorosomes in green sulfur bacteria is discussed based on the above results.