Influence of trans-membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells. Photoaffinity labelling of mitochondrial proteins, action of potential dissipating drugs, and competitive staining.

The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for photoaffinity labelling of mitochondrial proteins. By a combination of photoaffinity labelling cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonyl-cyanide-phenylhydrazone (CCCP), strongly reduces or even prevents APMC labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their Rm values in reversed phase thin-layer chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

The concentration jump method. Kinetics of vital staining of mitochondria in HeLa cells with lipophilic cationic fluorescent dyes.

Lipophilic cationic fluorescent dyes (D) specifically stain the mitochondria of living cells. A perfusion chamber for cell cultures is described, which can be used to determine the kinetics of vital staining of the mitochondria of single selected cells in situ. In these experiments styrylpyridinium dyes and cultures of HeLa cells were used. The dyes differ strongly in their lipophilic properties; Rm values and the partition coefficients Po/w between n-octanol (o) and water (w) were determined in order to characterize their lipophilicity. In the thermostat-regulated chamber the concentration of the dye CD can be increased from CD = 0 to CD > 0 within a few seconds (concentration jump). Thus, the time t = 0 for the beginning of the vital staining and the dye concentration in the cell medium during the staining experiment, CD = const., are unambiguously defined. The concentration of the dye, Cb, which is bound to the mitochondria (b), is proportional to the intensity of the fluorescence Ib. On the other hand, the free dye molecules (f) in the aqueous medium exhibit practically no fluorescence, I(f) << Ib. The intensity of the fluorescence I = Ib was measured as a function of time t; the measured values were corrected for photobleaching. The fluorescence intensity I(t) at first increases linearly with t and reaches a saturation value for t-->infinity. In the linear range of I(t) the flow J(o) = (dI/dt)o of the dye into the cell depends strongly on the dye concentration and increases linearly with CD. The concentration range CD = 10(-9)-10(-5) M at 37 degrees C was investigated. From the linear correlation between J(o) and CD it follows that the kinetics of the vital staining of mitochondria is controlled by diffusion. At t = 0 the flow of the xenobiotic agent through the cell membrane determines the rate of staining. The slope dJ(o)/dCD of the plot J(o) vs CD describes the efficiency of dye accumulation at the mitochondria and strongly increases with increasing lipophilicity of the dye molecules. Thus lipophilic dyes pass through the cell membrane more easily than less lipophilic molecules.

[The vital staining of nuclear chromatin and the chromosomes of HeLa cells with the fluorochrome AMHA binding to DNA]. / Uber die Vitalfluorochromierung des Kernchromatins und der Chromosomen von HeLa-Zellen mit AMHA und die Bindung des Acridinfarbstoffs an DNA.

The fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations CF less than or equal to 10(-4) M and short incubation periods tI less than or equal to 2 h cell growth is not affected by the drug. But at higher CF and longer tI the population doubling time of the cell cultures rapidly increases, and finally the cells die. In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm-1 between the 0-0-transitions of the long wave length 1La absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm-1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission. Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nucleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant KF1 = 1.1.10(5) M-1, binding parameter n1 = 0.15; the pre-intercalative or external binding 2, KF2 = 6.9.10(5) M-1, n2 = 0.21; the external binding 3, KF3 = 2.8.10(5) M-1, n3 = 0.55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant KF1 = 1.1.10(5) M-1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, KF1 = (1-4).10(4) M-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of ethidium bromide, tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ. A comparative binding study.

Several 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 approximately equal to 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.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hydrophobic acridine dyes for fluorescent staining of mitochondria in living cells. 3. Specific accumulation of the fluorescent dye NAO on the mitochondrial membranes in HeLa cells by hydrophobic interaction. Depression of respiratory activity, changes in the ultrastructure of mitochondria due to NAO. Increase of fluorescence in vital stained mitochondria in situ by irradiation]. / Uber hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen. 3. Mitteilung: Spezifische Akkumulation des Farbstoffs NAO an die Mitochondrienmembranen von HeLa-Zellen durch hydrophobe Wechselwirkung. Hemmung der Atmungsaktivität, Veränderung der Ultrastruktur der Mitochondrien durch NAO. Intensivierung der Fluoreszenz vital gefärbter Mitochondrien in situ bei Bestrahlen.

The hydrophobic fluorescence dye 10-n-nonyl-acridinium-orange-chloride, NAO, stains specifically the mitochondria of living HeLa-cells. A dye concentration of 1 X 10(-8) M is sufficient for vital staining and at 5 X 10(-7) M an incubation time less than 1 min is enough to generate the bright green fluorescence of the mitochondria. The retention of NAO by the mitochondria is longer than 7 days. The dye accumulation is not affected by the ionophores valinomycin, nigericin, gramicidin, the uncoupling agents DNP, CCCP or by ouabain. In contrast to Rh 123 the trans-membrane potential is not the driving force of the NAO accumulation. We assume that NAO is bound to the hydrophobic lipids and proteins in the mitochondrial membranes by hydrophobic interaction. With valinomycin, 500 ng/ml, 10 min, the mitochondria in HeLa-cells swell. Now it is possible to observe some details in the enlarged mitochondria by light microscopy. After vital staining with NAO, 5 X 10(-7) M, 10 min, the periphery of the swollen mitochondria shows an intense green fluorescence, the inner part is dark. Obviously the dye is bound to the membranes. By electron microscopy it can be shown that the valinomycin treated and NAO stained mitochondria have outer and inner membranes and cristae. They differ from untreated mitochondria mainly in the size. After incubation of the HeLa-cells with relatively high NAO concentrations, 5 X 10(-6) M, 10 min, the mitochondria show a weak orange fluorescence. It is generated by the dimers D of NAO. Therefore the dye concentration in the mitochondrial membranes is locally very high and causes dye dimerisation. The weak orange fluorescence is instable and disappears within a few seconds. Instead we observe a green fluorescence with growing intensity that is generated by the monomers M of NAO. The intensity has its maximum value after a few seconds. Using low NAO concentrations for incubation, 1 X 10(-7) M, 10 min, we observe only the green fluorescence with increasing intensity. In this case the orange fluorescence is too weak for observation (concentration quenching). It can be shown by experiments and quantum mechanics that the orange fluorescence is assigned to an optical forbidden, the green fluorescence to an allowed electronic transition of D or M respectively. Our results indicate a dissoziation of D in 2 M by irradiation of the mitochondria under the fluorescence microscope.(ABSTRACT TRUNCATED AT 400 WORDS)

[Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect]. / Uber Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt. 3. Mitteilung: Mikrospektralphotometrische Untersuchung der Romanowsky-Giemsa-Färbung. Spektroskopischer Nachweis eines DNA-Azur B-Eosin Y-Komplexes, der den Romanowsky-Giemsa-Effekt verursacht.

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)

[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]. / Uber hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen. 2. Mitteilung: Vergleich der Färbung lebender und fixierter HeLa-Zellen mit NAO und 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.

[Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine]. / Uber eine Bertalanffy-analoge Fluorochromierung mit 3-Dimethylamino-6-methoxyacridin.

Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS)

[The fluorescent staining of mitochondria in living HeLa- and LM-cells with new acridine dyes (author's transl)]. / Uber die Vitalfluorochromierung von Mitochondrien in HeLa- und LM-Zellen mit neuen Acridinfarbstoffen.

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.

[Fluorescent staining of chromosomes and nuclear structures in living LM- and HeLa-cells with new acridine dyes]. / Vitalfluorochromierung von Chromosomen und Kernstrukturen in LM- und HeLa-Zellen mit neuen Acridinfarbstoffen.

Fluorescent staining of chromosomes and nuclear structures (nucleolus associated chromatin) in living HeLa- and LM-cells (mouse fibroblasts) with new acridine dyes is reported. The dyes have aminoethylgroups in 9-position with different end groups at this residue (scheme of structures). Dyes without these 9-substituents only induce the formation of lysosomes. An exceptional position on vital staining of chromosomes and nuclear chromatin has the dye 3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine 1. Concentrations of 10(-3) M can be used in vital staining experiments. Measuring the consumption of oxygen we could demonstrate that the dye has no effect on the activity of respiration even at these high dye concentrations. Therefore we conclude that we have really observed vital staining and not postvital staining of chromosomes and nuclear chromatin. Similar properties has the well known vital dye acridine orange.

[On the quantitative fluorometric determination of cellular DNA by ethidium bromide and the constancy of the quantum yield of the ethidium DNA-complex in the biological environment (author's transl)]. / Uber die quantitative fluorometrische Bestimmung von DNA mit Ethidiumbromid in der Zelle und die Konstanz der Quantenausbeute des Ethidium-DNA-Komplexes im biologischen Milieu.

The fluorescent staining of fixed A9 cells (mouse fibroblasts) with ethidium bromide E has been investigated qualitatively and quantitatively. E is bound to DNA, RNA and protein. After enzymatic RNA digestion and staining at pH = 4.0 the dyestuff is bound to DNA specifically. The formed E-DNA-complex is stoichiometric if DNA will be saturated by highdye concentration, CF = 1.0 x 10(-2) M. The stoichiometric factor n = 0.21 was determined from Scatchard binding isotherms and by spectrophotometric titration. The binding of E to DNA and RNA has been investigated by binding isotherms. E is bound to DNA by intercalation and by ionic interaction. It is bound to RNA by ionic interaction only. The ionically bound dyestuff can be replaced by salts like LiCl from DNA and RNA. Only intercalated E remains. The competitive salt effect can be used to avoid the enzymatic RNA digestion in the specific staining of DNA. Within the cell the E-DNA-complex fluoresces strongly. Using a microspectrophotometer we determined the fluorescence intensity J and the extinction E in cell sections of q = 1 mu 2 area. The fluorescence was excited in the minimum of the absorbance of the E-DNA complex at lambda 1 = 365 nm. The extinction E2 was measured in the short wavelength band lambda 2 = 260 nm. Under these conditions we received a linear relation J = J (E2) up to high extinctions E2. The quantum yield Q of the E-DNA-complex is nearly constant and independent of the biological environment. Q is also unaltered by LiCl. Within the limits of error intercalated and ionic bound dye have the same quantum yeild. Under the conditions of stoichiometric staining the amount of E-DNA-complex and therefore DNA in the cell can be determined by J. The extinction coefficient epsilon 2 = 45 200 M-1 cm-1 of the E-DNA-complex at 260 nm was calculated from the concentration of dependence of the absorbance spectra using the law of mass action. By epsilon 2 the amount of DNA in the cell is accessible from J. According to our measurements with A9 cells the superficial density of DNA has the order of magnitude rho N = mN/q = 10(1) nmol cm-2, the amount mN of DNA per section 1 = 1 mu 2, mN = 10(-1) fmol, and the DNA concentration in the cell nucleus CN = 10(-1) M. The ray pass and diagram of electronics of the based microspectrophotometer are described.