H. W. Zimmermann

Selective photosensitization of mitochondria in HeLa cells by cationic Zn (II) phthalocyanines with lipophilic side-chains.

In order to optimize the photosensitization of mitochondria, we prepared the new cationic zinc (II) phthalocyanines with lipophilic side-chains (2,9,16,23-tetrakis (trimethylammonio) zinc (II) phthalocyanine tetramethylsulphate (ZnPcA1) and 2,9,16,23-tetrakis(hexyldimethylammonio) zinc (II) phthalocyanine tetramethylsulphate (ZnPcA6)) and compared them with the known cationic lipophilic sensitizers 2,9,16,23-tetrakis[2-(trimethylammonio) ethoxy]zinc (II) phthalocyanine tetraiodide (ZnPcE1) and 2,9,16,23-tetrakis[2-(hexyldimethylammonio) ethoxy] zinc (II) phthalocyanine tetrabromide (ZnPcE6) and the anionic hydrophilic sulphonated sensitizer ZnPcS. The absorption and fluorescence spectra, fluorescence quantum yields, partition coefficients Po/w between octanol and water, dimerization of ZnPcA1 in water and photosensitized generation of singlet oxygen were examined. Moreover, the phototoxic and cytotoxic effects of the phthalocyanines on HeLa cells, their photodynamic action on the proliferation rate and the influence of irradiation on the respiratory activity were studied and compared with electron and fluorescence microscopic observations of the ultrastructure of incubated and irradiated HeLa cells. From our experiments, we conclude that cationic lipophilic sensitizers, such as ZnPcA6, etc., specifically accumulate in the inner mitochondrial membranes. Therefore they can be used for the selective photosensitization of mitochondria. On irradiation of the incubated HeLa cells, the cristae are affected and finally completely destroyed. The respiration stops and the energy metabolism breaks down. Our experiments indicate that the phototoxic efficacy of the sensitizers decreases in the following order: ZnPcA6 > ZnPcA1 > ZnPcE6 > ZnPcE1 > ZnPcS. The cationic lipophilic ZnPcA6 has the strongest phototoxic activity. It acts on incubation at very low sensitizer concentrations and short irradiation periods. The dark toxicity is completely negligible. The phototoxic activity follows the type II mechanism.

[The fluorescent staining of mitochondria in living HeLa- and LM-cells with new acridine dyes (author's transl)].

SummaryThe fluorescent staining of mitochondria in living cells with new acridine dyes is reported. The fluorescent dyes used are derivatives of acridine orange (AO) and of 3-amino-6-methoxyacridine (AMA) with various residues in 9- or 10-position (Scheme 1). They are either permanent cationic dyes or cations which are formed by protonation in the culture medium. HeLa cells and mouse fibroblasts (LM cells) have been used for our staining experiments. On favourable conditions we succeeded in staining the mitochondria not only orthochromatically but also metachromatically. Photodynamical effects which have been observed during the exposure of the stained cells in the fluorescence microscope are described. The residues in 9- or 10-position favour the dye accumulation in the mitochondria. Vital staining with the basic compounds AO and AMA however leads to the formation of metachromatically stained lysosomes in the orthochromatically stained cytoplasm. The dye 3-amino-6-methoxy-9-(2-hydroxyethyl)acridine stains the nucleus of living cells.ZusammenfassungEs wird über die Fluorochromierung von Mitochondrien in lebenden Zellen mit neuen Acridinfarbstoffen berichtet. Die verwendeten Fluorochrome sind Derivate von Acridinorange (AO) und von 3-Amino-6-methoxyacridin (AMA) mit verschiedenen Resten in 9- bzw. 10-Stellung (Formelschema 1). Sie sind entweder permanente Farbstoffkationen oder liegen im Kulturmedium als Kationen vor. HeLa-Zellen und Mäusefibroblasten (LM-Zellen) wurden fluorochromiert. Unter günstigen Bedingungen gelang es, die Mitochondrien nicht nur orthochromatisch sondern auch metachromatisch zu färben. Über photodynamische Effekte, die bei der Bestrahlung unter dem Fluoreszenzmikroskop auftreten, wird berichtet. Die Reste in 9- bzw. 10-Stellung begünstigen die Farbstoffakkumulation in den Mitochondrien. vitalfärbung mit den Grundkörpern AO bzw. AMA ergibt demgegenüber metachromatisch gefärbte Lysosomen im orthochromatisch gefärbten Cytoplasma. Der Farbstoff 3-Amino-6-methoxy-9-(2-hydroxyethyl)-acridin fluorochromiert den Kern lebender Zellen.The fluorescent staining of mitochondria in living cells with new acridine dyes is reported. The fluorescent dyes used are derivatives of acridine orange (AO) and of 3-amino-6-methoxyacridine (AMA) with various residues in 9- or 10-position (Scheme 1). They are either permanent cationic dyes or cations which are formed by protonation in the culture medium. HeLa cells and mouse fibroblasts (LM cells) have been used for our staining experiments. On favourable conditions we succeeded in staining the mitochondria not only orthochromatically but also metachromatically. Photodynamical effects which have been observed during the exposure of the stained cells in the fluorescence microscope are described. The residues in 9- or 10-position favour the dye accumulation in the mitochondria. Vital staining with the basic compounds AO and AMA however leads to the formation of metachromatically stained lysosomes in the orthochromatically stained cytoplasm. The dye 3-amino-6-methoxy-9-(2-hydroxyethyl)acridine stains the nucleus of living cells.

New investigations on hematoxylin, hematein, and hematein-aluminium complexes

SummaryAnalytically pure hematoxylin (Htx), penta-acetylhematoxylin (PAHtx), and hematein (Hm) were isolated and characterized by 1H-NMR spectroscopy. The VIS/UV spectra of Htx and Hm were recorded in MeOH and in H2O at various pH values. The molar extinction coefficients of the long wavelength absorption bands are reported. The pKa value for the 1st acidic dissociation step of Hm has been determined from the pH dependency of the absorption spectra of Hm in aqueous buffer solutions. Finally, the absorption spectra are qualitatively discussed.

Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt

SummaryThe Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed.The nucleus of RG stained LM cells, pH≃7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates.In the spectrum of RG stained nuclei, pH≃7, three absorption bands are distinguishable: They are A1 (15400 cm−1, 649 nm), A2 (16800 cm−1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm−1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of assoziation p>2) and eosin Y. The complex is primarily held together by electrostatic interaction: Binding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both moleculare species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm−1 is due to the cosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei.Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y. Its absorption spectrum is identical with the spectrum of Romanowsky stained nuclei. This experiment demonstrates that the whole Romanowsky-Giemsa staining pattern of the nucleus is primarily produced by DNA, azure B and eosin Y.The spectrum of RG stained cytoplasm, pH≃7, consists of three absorption bands A1′ (15300 cm−1, 654 nm), A2′ (16900 cm−1, 592 nm), A3′ (17600 cm−1, 568 nm). They are attributed to monomers, dimers and polymers of azure B bound to RNA. In general A2′ and A3′ overlap strongly and generate a broad absorption band A′ (∼17000 cm−1, 588 nm). Some experimental results can be interpreted in terms of a RNA-azure B-eosin Y complex in the cytoplasm. But its concentration must be very small.Variations in the biological materials and the experimental staining conditions may alter the position and the intensity of the absorption bands but does not change the underlying molecular concept of the Romanowsky-Giemsa effect.The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)

ber hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen@@@Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells: 1. Mitteilung: Thermodynamische und spektroskopische Eigenschaften von 10-n-Alkyl-acridiniumorange-chloriden@@@1. Thermodynamic and spectroscopic properties of 10-n-alkyl-acridinium-orange-chlorides

10-n-Alkyl-acridine-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl = methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K-1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K = K0 at the standard temperature T = 298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m + 1 (m = 1 - 9) (Table 2). In addition the standard dissociation enthalpies (energies) delta H0 and dissociation entropies delta S0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K0, delta H0, delta S0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M1 = 20.000 cm-1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm-1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D1 = 18.700 and D2 = 21.200 cm-1 with very different intensities. D1 has very low intensity and is forbitten, D2 is allowed. D1 shows fluorescence. Phosphorescence has not been observed. D1, D2 and also M1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition iSummary10-n-Alkyl-acridinium-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl=methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K−1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K=K0at the standard temperature T=298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m+1 (m=1–9) (Table 2). In addition the standard dissociation enthalpies (energies) Δ H0 and dissociation entropies Δ S0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K0, Δ H0, Δ S0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M1=20.000 cm−1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm−1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D1=18.700 and D2=21.200 cm−1 with very different intensities. D1 has very low intensity and is forbitten, D2 is allowed. D1 shows fluorescence. Phosphorescence has not been observed. D1, D2and also M1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition intensities agree with quantum mechanical model calculations for dimers with parallel or antiparallel molecular orientation. Hydrophobic interaction needs parallel orientation in the dimers of nonyl-AO. In the dimers of AO and of dyes with very short alkyl chains antiparallel orientation may occur.

[Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 2. Comparison of staining of living and fixed Hela-cells with NAO and DPPAO].

The hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithin-like dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occurs even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.SummaryThe hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithinlike dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occures even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.

TIME-RESOLVED MICROFLUOROMETRIC STUDY OF THE BINDING SITES OF LIPOPHILIC CATIONIC PYRENE PROBES IN MITOCHONDRIA OF LIVING HELA CELLS

Lipophilic dye cations specifically bind to the mitochondria of living cells. Using fluorescent dyes, the mitochondria can easily be observed with a fluorescence microscope. Electron microscopy has shown that the dyes are bound to the inner mitochondrial membranes and the cristae. Using time-resolved fluorescence microscopy we have investigated, whether the dye molecules are preferentially accumulated at the strongly hydrophobic protein complexes of energy metabolism or at the lipids of the inner membrane system. In order to use our nanosecond-pulsed laser fluorometer we synthesized specially designed lipophilic pyrene cations with S1 lifetimes in the nanosecond domain, which specifically stain mitochondria in living HeLa cells. Model experiments with artificial membranes such as liposomes, proteoliposomes and also protein complexes have shown that the fluorescence is strongly quenched by oxygen if the pyrene probes are bound to lipids. Binding to proteins causes a much smaller quenching effect. In artificial systems, all decays were single exponential. This is in contrast with incubated HeLa cells, which showed double-exponential fluorescence decays. Comparing these with the artificial systems we came to the conclusion that in HeLa cells the long-lived species 1 are pyrene probes preferentially bound to the proteins of the inner mitochondrial membranes. The short-lived species 2 is caused by fluorescence resonance energy transfer from the pyrene probes as donors to cytochromes of the inner membranes as acceptors. From our decay data we estimated a mean distance between donor and acceptor of about 40 A. This is the same order of magnitude as the mean diameters of several mitochondrial protein complexes. Therefore we assumed that species 2 are pyrene probes bound either to mitochondrial proteins with cytochromes as constituents or to the interface between these proteins and the phospholipids of the membranes. Thus both species 1 and species 2 are spatially related to mitochrondrial proteins. This agrees with the observation that respiration of HeLa cells as well as cytochrome c oxidase vesicles (COVs) are inhibited with increasing concentration of pyrene probes. Finally, we studied the photodynamic effect on irradiation of HeLa cells and of COVs after incubation with lipophilic pyrene and porphyrine cations.

Effects of ethidium bromide, tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ

SummarySeveral investigators have described the ultrastructural changes that occur in the mitochondria of cells in tissue cultures after treatment with the drug ethidium bromide (E). The mitochondria swell and the cristae become greatly altered and finally disappear; in the cristae-free region of the matrix electron-dense granules can be observed. It has been assumed that intercalation of E between the base pairs of the mitochondrial DNA induces the formation of the granular inclusions. To investigate whether intercalation is really the initial step in the generation of dense granules inside the matrix, we performed a comparative incubation study of HeLa-cell mitochondria in situ using three closely related dyes (D), i.e. E, tetramethylethidium bromide (TME) and betaine B (B). They strongly differ with regard to their affinity for DNA and their ability to cross membranes. E was used as a reference dye. TME does not intercalate, but is externally bound to DNA only weakly. The neutral B is not bound at all, but can cross membranes more easily than the cation E. Moreover, in aqueous solutions at pH≃7.0, B is in equilibrium with its protonated cation BH. BH and E have almost equal affinities for DNA. Therefore B may quickly pass the inner mitochondrial membranes and the cristae, and should then be bound inside the matrix, thus forming a BH-DNA complex. On the assumption that intercalation is necessary for the generation of intramitochondrial electron-dense bodies, we predicted that BH/B should be more efficient than E, while TME should be relatively ineffective. In experiments using HeLa cells, these predictions were found to be inaccurate. E, TME and BH/B produced almost the same mitochondrial alterations, but at different concentrations and after different incubation periods. In contrast to our expectations TME was much more effective than E and BH/B, with the last two behaving rather similarly.Therefore, it seems unlikely that the drugs penetrate the inner mitochondrial membrane system by simple physical diffusion or that intercalation is the preliminary step for the generation of dense granules inside the matrix. Instead, we assume that hydrophobic interaction between the dye cations E, BH and TME and the cristae is the main cause of the mitochondrial changes. The favoured binding partner of the dye cations may be the divalent anion, cardiolipin: this phospholipid is an essential part of the inner membrane system but is absent in other membranes of cells. By distributing the dyes between a lipophilic phase and water, it was shown that TME is more lipophilic than E and BH; this may explain the greater effectiveness of TME. The bound dye cations disturb the organization of the cristae, which become altered and finally disappear. We assume that the electron-dense granules in the matrix are mainly composed of the dyes and former membrane materials such as phospholipids and proteins, as well as perhaps some other hydrophobic matrix materials. This would also explain why it was impossible to digest the dense granules by DNase treatment. The drugs enter the mitochondrial matrix by disordering and finally destroying the cristae.

Selective photosensitization of mitochondria by the lipophilic cationic porphyrin POR10

The lipophilic cationic porphyrin derivative 5.10.15.20-tetrakis(1-decylpyridinium-4-yl)-21H.23H-++ +porphin tetrabromide (POR10) binds to the mitochondria of living HeLa cells and can be used for the selective photosensitization of mitochondria. Cytotoxic and phototoxic effects were studied by means of electron microscopy and respiratory activity and compared with the effects of the hydrophilic cationic 5.10.15.20-tetrakis(1-methylpyridinium-4-yl)-21H.23H-porp hin tetraiodide (POR1) and the anionic 5.10.15.20-tetrakis(1-sulphophen-4-yl)-21H,23H-porp hin (TPPS). Lipophilic and hydrophilic molecules behave quite differently. The mitochondria are much more attacked by the lipophilic POR10 than by the hydrophilic POR1 and TPPS. At low POR10 concentrations and short incubation periods (C(D) < 0.1 microM; tI < 1 h), the mitochondrial ultrastructure scarcely alters, but, on increasing concentrations and prolonged incubation periods, in particular the cristae were affected. Irradiation (lambda > 425 nm) of incubated HeLa cells also strongly attacks the cristae. For example, after incubation under mild conditions (0.1 microM; 1 h) and subsequent irradiation (12 min at 65 mW cm(-2)) the cristae were partially destroyed. After incubation under intensified conditions (1 microM; 1 h) and subsequent irradiation (15 min at 25 mW cm(-2)) the cristae disappeared completely and the mitochondria were transformed into globular vesicles surrounded by a double membrane. Surprisingly, respiratory activity increased (115%) after POR10 treatment under mild conditions (0.1 microM; 30 min). However, subsequent irradiation for only 2 min (65 mW cm(-2)) decreased the respiratory activity to 18% relative to the untreated control (100%). Much smaller effects were observed with POR1-treated cells. We assume that the lipophilic POR10 cations, like other lipophilic dye cations, bind to the very lipophilic enzymes of the respiratory chain, which are constituents of the cristae. In contrast with POR10 the hydrophilic porphyrin derivatives are spread over several cellular compartments and are less phototoxic.

Spektroskopische und thermodynamische Untersuchungen zur Bindung von Azur B an Chondroitinsulfat und zur Bindungsgeometrie des metachromatischen Farbstoffkomplexes

The binding of azur B to chondroitin sulfate (CHS) was investigated using absorption spectroscopy. In aqueous solutions it is possible to distinguish three different dye species with absorption bands at 646, 597, and 555 nm. They are assigned to monomers, dimers, and higher aggregates of azure B, which become bound to CHS as the dye concentration (CD) increases. The short-wavelength band (555 nm) causes metachromasia in stained histological materials. When saturation occurs, the metachromatic azure B-CHS complex has a 1:1 composition, i.e., each anionic SO-4 and COO(-)-binding site of CHS binds one dye cation. The composition of the saturated metachromatic complex was determined by spectrophotometric and conductometric titration of CHS with azure B, while the SO-4 and COO- content of CHS was determined by conductometric titration of CHS-acid with NaOH. The binding isotherm of azure B to CHS was determined using gelpermeation chromatography. The isotherm can be described by the model of cooperative binding of ligands to linear biopolymers. We found good agreement between theoretical predictions and experimental findings in the range of 0 less than r less than 0.8 (r = the fraction of occupied binding sites). Using a Schwarz plot, we determined the binding constants of nucleation (Kn = 2.5 X 10(3) M-1) and aggregation (Kq = 1.2 X 10(5) M-1), as well as the cooperativity parameter (q = 50), T = 295 K. With increasing CD, the strong cooperativity of the dye binding favors the formation of metachromatic aggregates rather than monomers and dimers. From the temperature dependence of Kq we evaluated the standard binding enthalpy (delta Hoq = -20.0 kJ mol-1) and entropy (delta Soq = 29.7 JK-1 mol-1) of the cooperative dye binding. The binding was found to be strongly exothermic and accompanied by a thermodynamically favorable entropy increase, this being typical of hydrophobic interactions. Solid azure B-CHS complexes were prepared according to a special dialytic technique and were studied using a microspectrophotometer equipped with a polarizer and an analyzer. The metachromatic 1:1 complex has a broad, intense absorption band whose main peak occurs at 560 nm. This corresponds with the maximum of the metachromatic dye complex in aqueous solution, i.e. 555 nm. The CHS chains of the azure B-CHS complex can be mechanically aligned in a preferred direction (k). We were able to prepare excellently orientated and very fine dye-CHS films which were birefringent and dichroic - the more birefringent, the better the mechanical orientation.(ABSTRACT TRUNCATED AT 400 WORDS)SummaryThe binding of azur B to chondroitin sulfate (CHS) was investigated using absorption spectroscopy. In aqueous solutions it is possible to distinguish three different dye species with absorption bands at 646, 597, and 555 nm. They are assigned to monomers, dimers, and higher aggregates of azure B, which become bound to CHS as the dye concentration (CD) increases. The short-wavelength band (555 nm) causes metachromasia in stained histological materials. When saturation occurs, the metachromatic azure B-CHS complex has a 1:1 composition, i.e., each anionic SO−4-and COO−-binding site of CHS binds one dye cation. The composition of the saturated metachromatic complex was determined by spectrophotometric and conductometric fitration of CHS with azure B, while the SO−4and COO− content of CHS was determined by conductometric titration of CHS-acid with NaOH.The binding isotherm of azure B to CHS was determined using gelpermeation chromatography. The isotherm can be described by the model of cooperative binding of ligands to linear biopolymers. We found good agreement between theoretical predictions and experimental findings in the range of 0<r<0.8 (r=the fraction of occupied binding sites). Using a Schwarz plot, we determined the binding constants of nucleation (Kn=2.5·103 M−1) and aggregation (Kq=1.2·105 M−1), as well as the cooperativity parameter (q=50), T=295 K. With increasing CD, the strong cooperativity of the dye binding favors the formation of metachromatic aggregates rather than monomers and dimers. From the temperature dependence of Kq we evaluated the standard binding enthalpy (†Hqo=20.0 kJ mol−1) and entropy (†Sqo=29.7 JK−1 mol−1) of the cooperative dye binding. The binding was found to be strongly exothermic and accompanied by a thermodynamically favorable entropy increase, this being typical of hydrophobic interactions.Solid azure B-CHS complexes were prepared according to a special dialytic technique and were studied using a microspectrophotometer equipped with a polarizer and an analyzer. The metachromatic 1:1 complex has a broad, intense absorption band whose main peak occurs at 560 nm. This corresponds with the maximum of the metachromatic dye complex in aqueous solution, i.e. 555 nm. The CHS chains of the azure B-CHS complex can be mechanically aligned in a preferred direction (k). We were able to prepare excellently orientated and very fine dye-CHS films which were birefringent and dichroic-the more birefringent, the better the mechanical orientation. The site of best orientation within the preparations was selected according to the quality of the birefringence, and this region was then measured with a microspectrophotometer using linearly polarized light. By setting the polarizer (ep) parallel and perpendicular to k, we obtained the dichroic ratio (d) of the light absorbance (E, d=E∥/E⊥). A dichroic ratio of d=0.1 was determined for the 560 nm absorption band of the metachromatic dye complex. Therefore, the transition moment (m) of the dye molecules is polarized almost perpendicularly to the preferred direction, k, m⊥k. The moment (m) of the