Quantitative Bestimmung von Sulfhydrylgruppen mit “Mercurochrom”

Zusammenfassung Dibrommerkurifluoreszein (DBMF) reagiert stöchiometrisch und quantitativ mit der SH-Gruppe von Cystein, Glutathion und Thioglycolsäure. Polarographische und spektrometrische Titrationen ergeben, daß die Wellenlänge des ersten Absorptionsmaximums von DBMF (507 nm) bei den gebildeten Merkaptiden unverändert bleibt, während der molare Extinktionskoeffizient um rund 20% ansteigt. Serumalbumin, Ovalbumin, -Laktoglobulin und Glycerinaldehydphosphat-Dehydrogenase bilden nach Inkubation mit DBMF Addukte aus denen die reinen DBMF-Protein-Merkaptidkomplexe säulenchromatographisch isoliert wurden. Sie zeigen im Absorptionsspektrum eine bathochrome Verschiebung der Farbstoffbande (520 nm) mit um ca. 50% erniedrigtem Extinktionskoeffizienten (₅₂₀=32000–33850). Die mit diesem Wert aus den Maximumsextinktionen berechneten SH-Gehalte entsprechen den auf Grund von Literaturangaben zu erwartenden Daten. Eine selektive Reaktion, z.B. mit besonders zugänglichen oder hoch-reaktiven SH-Gruppen, konnte mit DBMF nicht festgestellt werden. Native tierische Tumorzellen zeigen nach 30 min Inkubation mit DBMF und Auswaschen mit isotonem Phosphat-Puffer in Kern und Plasma als Hauptbande das rotverschobene Maximum, an dem jedoch auch das unverschobene Maximum als mehr oder minder deutliche Inflexion beteiligt ist. Probeweise, mit ₅₂₀ ausgeführte Berechnungen des Protein-SH-Gehaltes zeigten 1,7–2,1·10^–14 Mole/Zelle an. Dieses vorläufige Ergebnis liegt zwischen den in früheren eingehenden Untersuchungen mit DDD-Echtblau mikrospektrometrisch (1,1–1,55·10^–14) und mit DTNB makroskopisch gefundenen SH-Gehalten von EATZ (3,1·10^–14). Ob und wie stark bei den mit DBMF gefundenen Werten auch unspezifische Adsorption beteiligt ist, läßt sich gegenwärtig noch nicht sicher beurteilen. Eine Reaktion mit Nukleinsäure konnte auf Grund von Modellversuchen jedoch mit Sicherheit ausgeschlossen werden.

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Quantitative determination of sulfhydryl groups with mercurochrome

Summary Dibrommercuryfluoresceine (DBMF) reacts stoichiometrically and quantitatively with the thiol group of cysteine, glutathione and thioglycolic acid respectively, at pH 7.0. Polarographical and spectrometrical titrations clearly show that in the spectra of the investigated mercaptides the wave length of the first absorption maximum of DBMF (507 nm) remains unchanged but the molar extinction coefficient increases by approximately 20%. Serum albumin, ovalbumin, -lactoglobulin and glyceraldehydephosphatedi-hydrogenase after incubation with DBMF, form adducts with the dye from which the pure mercaptide complexes were separated by means of column chromatography. These complexes show a bathochromic shift (520 nm) of the dye band which is decreased now by 50%. The molar extinction coefficient ₅₂₀ has been determined from 32,000 to 33,850. On the basis of these values SH-contents of the four proteins were obtained which are in good accordance with data previously published in the literature. No selective reaction, f.i. with more accessible or/and reactive SH-groups was observed. After 30 min incubation with DBMF and washing with isotonic phosphate buffer, native animal tumor cells show in the main absorption band the bathochromically shifted dye maximum. A first temptative estimation of the protein SH-groups yielded 1.7–2.1×10^–14 mole SH/single cell. This result lies between the SH-content determined microspectrometrically on cells stained with DDD-Fast Blue B (1.1–1.55×10^–14) and macroscopically on cell homogenates with DTNB (3.1×10^–14). Up to now, no certain information can be given whether or to what extent unspecific absorption effects possibly might be involved in the data obtained with DBMF treated cells, but interaction with nucleic acids can be excluded with certainty on the basis of relevant model experiments.

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Diese Arbeit wurde mit Unterstützung des Fonds zur Förderung der wissenschaftlichen Forschung, Wien, durchgeführt

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Herrn Prof. Dr. G. Zigeuner zum 60. Geburtstag gewidmet.     

Quantitative Bestimmung von Sulfhydrylgruppen mit Mercurochrom

Zusammenfassung Native Ehrlich-Ascites Tumorzellen (EATZ) werden in einer 1×10^–3 M Lösung von Dibrommercurifluoreszein (DBMF) in Sörensen-Phosphatpuffer pH 6.7+0,9% NaCl 30 min inkubiert und sodann viermal mit dem gleichen Puffer, der 0,01 M in bezug auf NaCN ist, bis zur Farblosigkeit des Überstandes gewaschen. Die Zellen zeigen nun ein Absorptionsmaximum zwischen 520 und 525 nm, das mit dem von Komplexen reiner Korpuskularproteine mit DBMF identisch ist. Die Zellen werden im Scanning bei 520 nm photometriert und daraus ihre Gesamtextinktionen ermittelt. Unter Zugrundelegung des in früheren Arbeiten bei den Protein-DBMF-Komplexen bestimmten Extinktions-koeffizienten =33 000 ergibt sich ein Gehalt von rund 1,1×10^–14 Molen Proteinthiolen (Prot-SH) pro Zelle. Dieser Wert entspricht sowohl dem von Nöhammer 1982 mit Dihydroxydinaphthyldisulfid gefundenen Gehalt an reaktiven, d.h. schnell reagierenden Prot-SH, als auch dem von Rindler et al. 1970 bestimmten SH-Gehalt der primär löslichen Zellproteine. Da aus nativ mit DBMF behandelten Zellen jedoch keine im Homogenat-Puffer löslichen Proteine gefunden wurden, ist es wahrscheinlich, daß DBMF mit den schnellen SH-Gruppen der Cytosol-Proteine reagiert, die dabei strukturelle Veränderungen erfahren, die zum Löslichkeitsverlust führen. DBMF erfaßt jedoch die gesamten Prot-SH, reaktive und maskierte, wenn es auf vorher fixierte Zellen einwirkt (Nöhammer et al. 1981). Ist der zum Waschen verwendete Puffer Cyanid-frei, so wird die identische Absorptionsbande gemessen; die für E _tot,520 ermittelten Werte sind jedoch um 60% höher. Die Differenz wird nicht-kovalent, reversibel an die Zellen gebundenem DBMF zugeschrieben.

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Quantitative determination of sulfhydryl groups with MercurochromeII. Detection of fast reacting protein thiols in native cells

Summary Native Ehrlich ascites tumor cells (EATC) are incubated during 30 min in a 1×10^–3 M solution of dibrommercurifluoresceine (DBMF) in Sörensen phosphate buffer pH 6.7+0.9% NaCl. Subsequently the cells are washed four times in the same buffer containing additionally 0.01 M NaCN until the supernate appears to be colourless. They show an absorption maximum between 520 and 525 nm which is identical with that of complexes between pure corpuscular proteins and DBMF, investigated previously. Scanning at 520 nm yields the total extinction of the stained cells E _tot which is calculated into moles of protein bound thiol groups (prot-SH), with an extinction coefficient =33,000 previously determined with the protein-DBMF-complexes. A mean protein-SH content of 1.1×10^–14 moles per single cell is found which corresponds with both the content of fast reacting prot-SH previously determined by Nöhammer with dihydroxydinaphthyldisulfide, and the SH-content of the soluble cellular proteins determined by Rindler et al. with DTNB. As no soluble proteins could be obtained from DBMF-treated cells, it can be assumed that in native cells DBMF reacts preferentially with the fast reacting SH-groups of the soluble proteins which, in the course of structural changes, became insoluble. According to Nöhammer et al. (1981), however, DBMF also reacts with the total of protein-SH content of 1.1×10^–14 moles per single cell is found which fixed with ethanol/ether. When the buffer used for washing is free from CN^–, the identical absorption band is measured; however, the values determined for E _tot,520 are approximately 60% higher. The extinction difference is ascribed to DBMF, noncovalently and reversibly bound to the cells.

     

Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, erythrosin B, tetrachlorofluorescein, spectroscopic characterization of pure dyes, association of eosin Y

Analytically pure samples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependent on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimers D in monomers M at 293 K, pH = 12, is K21 = 2,9 X 10(-5) M; of the tetramers Q in dimers D K42 = 2,4 X 10(-3) M. From the experimental spectra of eosin solutions at various concentrations, pH = 12, and the equilibrium constants K21, K42 the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, VM = 19300 cm-1, epsilon M = 1,03 X 10(5) M-1 cm-1; D also one absorption band, VD = 19300 cm-1, epsilon D = 1,74 X 10(5) M-1 cm-1; Q two absorption bands, VQ1 = 19100, VQ2 = 20200 cm-1, epsilon Q1 = 1,65 X 10(5), epsilon Q2 = 1,96 X 10(5) M-1 cm-1. The absorption spectrum of the dimers is discussed by quantum mechanics.     

The metallochromic indicator dye Arsenazo III forms 1:1 complexes with caffeine

The metallochromic indicator dye, Arsenazo III, forms a 1:1 complex with caffeine, a methylxanthine. Binding is accompanied by a wavelength-dependent shift in the absorption spectrum of the dye. The magnitude of the absorption change is significant at wavelengths typically used to monitor intracellular calcium ion. The equilibrium constant for the caffeine-dye reaction is approx. 20 mM. The complex has a differential molar extinction coefficient of -5.05 X 10(3) M-1 X cm-1 at 630 nm.     

New investigations on hematoxylin, hematein, and hematein-aluminium complexes. II. Hematein-aluminium complexes and hemalum staining.

The absorption spectra of hematein-aluminium solutions have been recorded at various concentrations and pH values; the solutions were prepared using analytically pure hematein and potassium alum as aluminium source. In aqueous solution, four different hematein-aluminium complexes could be distinguished by absorption spectroscopy. In weakly acidic media we observed the violet 1:1 and 1:2 complexes HmAl (VII) and HmAl2(3) (VIII), and in strongly acidic solution the red 1:1 complex HmAl2 (IX). Whereas, in weakly alkaline solution the blue 1:1 complex HmAl0 (X) was detected. By change of the pH value the complexes were mutual interconverted. The dye complexes were characterized by their absorption spectra and molar extinction coefficients. We have stained HeLa cells with the complex solutions under different experimental conditions. In all cases the nuclear staining was intense whereas the staining of the cytoplasm was weak. The microspectra of the stained nuclei were recorded and compared with the absorption spectra of the complexes in solution. Thus it was possible to identify the bound dye species. After staining in acidic media, the cells were red to red-violet depending on the reaction conditions. The three cationic dye species VII, VIII, and IX were bound in varying amounts. After blueing in weakly acidic media or in water, only the violet dye complex VII was detected whereas, after blueing in weakly alkaline media, only the blue complex X has been observed. Enzymatic digestion experiments have shown that the dye complexes in the nuclei were bound to DNA while those in the cytoplasm and nucleoli were bound to RNA. The binding between the dye complexes and the nucleic acids is discussed.     

Interactions of pyronin Y(G) with nucleic acids

Spectral properties of pyronin Y(PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 X 10(3), M-1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single-stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double-stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 X 10(4), M-1) and to ds DNA (Ki = 1.74 X 10(4), M-1) was measured in 150 mM NaCl; the binding site size was 2-3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.     

Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina

11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X [3H]11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark.     

Mechanism of interaction between milk xanthine oxidase and p-chloromercuribenzoate. Properties of the purified enzyme]

Two types of complexes are formed during the interaction of xanthine oxidase with p-chloromercurybenzoate (pCMB). The reversible inactive complex (presumably of absorption nature) is formed practically instantaneously and competitively with regard to the substrate (Ki=6,2 . 10(-8) M) in 0,05 M phosphate buffer (pH 7,8, 25 degrees) and does not involve the fast-reacting SH-groups of the enzyme. Reactivation of xanthine oxidase is observed during prolonged incubation of the inactive complex at 0 degrees; it is associated with the interaction between pCMB and the fact-reacting SH-groups. This interaction results in a dissociation of the inactive complex. The blocking of the slow-reacting SH-groups is accompanied by an irreversible loss of the xanthine oxidase activity. The enzyme modification by blocking of 10 fast-reacting SH-groups does not involve the Fe-S clusters, but results in local changes in the enzyme conformation. This is manifested in a 2-fold increase of Km and the rate constants of proteolysis of the modified xanthine oxidase as compared to the native enzyme. The rate constants of proteolysis by trypsin for the native and modified enzymes in 0,05 M phosphate buffer (pH 7,8; 37 degrees) are 3,7 . 10(-3) min-1 and 7,0 . 10(-3) min-1, respectively; those for chymotrypsin in the same buffer (30 degrees) are 1,5 . 10(-2) min-1 and 6,0 . 10(-2) min-1, respectively.     

Reaction of lecithin:cholesterol acyltransferase with micellar complexes of apolipoprotein A-I and phosphatidylcholine, containing variable amounts of cholesterol

In a continued investigation of lecithin:cholesterol acyltransferase reaction with micellar, discoidal complexes of phosphatidylcholine (PC) . cholesterol . apolipoprotein A-I (apo-A-I), we prepared well defined complexes with variable free cholesterol contents and examined their reactivity with purified enzyme. The complexes, prepared by the sodium cholate dialysis method, were fractionated into "small" and "large" classes by gel filtration of the reaction mixtures through a Bio-Gel A-5m column. The small complexes had egg-PC/cholesterol/apo-A-I molar ratios from 68:14:1 to 80:1:1, discoidal shapes with diameters around 114 (+/- 13) A and widths of 42 A by electron microscopy, and Stokes radii from 47 to 49 A corresponding to molecular weights near 2 X 10(5). The corresponding properties of the large complexes, isolated from samples with higher cholesterol contents, were egg-PC/cholesterol/apo-A-I molar ratios from 84:26:1 to 96:17:1, diameters of 161 (+/- 20) A, widths of 43 A, Stokes radii around 80 A, and estimated molecular weights in the vicinity of 5 X 10(5). Both types of complexes, when adjusted to equal apo-A-I concentrations, gave essentially identical initial reaction velocities with purified lecithin:cholesterol acyltransferase over a wide range of cholesterol concentrations (from 2 X 10(-7) to 4 X 10(-4) M), PC/cholesterol molar ratios (from 3:1 to 12:1), and quite different lipid fluidity conditions as detected by diphenylhexatriene fluorescence polarization. When complexes were adjusted to a constant cholesterol concentration, the initial velocities of the lecithin:cholesterol acyltransferase reaction followed Michaelis-Menten kinetics relative to the apo-A-I concentrations. Arrhenius plots of initial reaction rates for various complexes with variable cholesterol content and fluidity, measured at constant apo-A-I concentrations, gave identical temperature dependences with an average activation energy of 18.0 kcal/mol. These results strongly suggest that the cholesterol esterification on high density lipoprotein particles does not depend on their unesterified-cholesterol contents, PC/unesterified-cholesterol molar ratios, nor on the fluidity of their lipid domains.     

Romanowsky dyes and romanowsky-Giemsa effect. 1. Azure B, purity and content of dye samples, association (author's transl)

Azure B is the most important Romanowsky dye. In combination with eosin Y it produces the well known Romanowsky-Giemsa staining pattern on the cell. Usually commercial azure B is strongly contaminated. We prepared a sample of azure B-BF4 which was analytically pure and had no coloured impurities. The substance was used to redetermine the molar extinction coefficient epsilon (v)M of monomeric azur B in alcoholic solution. In the maximum of the long wavelength absorption at v = 15.61 kK (lambda = 641 nm) the absorptivity is epsilon (15.61)M = (9.40 +/- 0.15) x 10(4)M-1 cm-1. This extinction coefficient may be used for standardization of dye samples. In aqeuous solution azur B forms dimers and even higher polymers with increasing concentration. The dissociation constant of the dimers, K = 2,2 x 10(-4)M (293 K), and the absorption spectra of pure monomers and dimers in water have been calculated from the concentration dependence of the spectra using an iterative procedure. The molar extinction coefficient of the monomers at 15.47 kK (646 nm) is epsilon (15.47)M = 7.4 x 10(4)M-1 cm-1. The dimers have two long wavelength absorption bands at 14.60 and 16.80 kK (685 and 595 nm) with very different intensities 2 x 10(4) and 13.5 x 10(4)M-1 cm-1. The spectrum of the dimers in aqueous solution is in agreement with theoretical considerations of F?rster (1946) and Levinson et al. (1957). It agrees with an antiparallel orientation of the molecules in the dimers. It may be that dimers bound to a substrate in the cell have another geometry than dimers in solution. In this case the weak long wavelength absorption of the dimers can increase.     

Receptor-mediated degradation by rat adipocytes: comparison of A-14 with A-19 125I-labelled insulin

We compared A-14 and A-19 125I-labelled insulin in receptor-binding and degradation. Percent receptor-binding of A-14 and A-19 125I-labelled insulin to 2.4 X 10(9)/ml erythrocytes after 210 min incubation at 15 degrees C was 7.8 and 4.9%, respectively. Percent insulin-receptor binding of A-14 insulin was 1.6 times greater than that of A-19 insulin. A similar result was obtained in an adipocytes insulin binding study. Percent receptor-binding of A-14 and A-19 insulin to 2 X 10(5)/ml fat cells after 30 min incubation in the above buffer was 3.9 and 2.4%, respectively. Degradation of A-14 and A-19 insulin in rat adipocytes was also studied by molecular sieve column chromatography. Isolated rat adipocytes were allowed to associate with A-14 and A-19 125I-insulin for 60 min at 37 degrees C, pH 8.0 in a HEPES-phosphate buffer, and then cells were separated from the buffer by centrifugation. After solubilization with triton X-100, both the solubilized cells and the incubation medium were applied to the Bio-Gel P-30 column to assess the insulin degradation. Degradation of A-14 125I-insulin by the isolated rat adipocytes was 1.6 times greater than that of A-19 125I-insulin. Furthermore, the peak which was thought to be intermediate degradation products of insulin was obtained between the peak of intact insulin and that of 125I-tyrosine. Such a peak of intermediates was much smaller in the incubation media than in the cell-associated materials.(ABSTRACT TRUNCATED AT 250 WORDS)     

A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate

A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions.     

Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine

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)     

Photocytotoxicity of anthracyclines upon laser excitation in their long-wavelength absorption bands

Newly synthesized daunomycin derivatives with red-shifted absorption compared to the parent molecule are shown to be able to photosensitize cells in vitro upon excitation with either argon or argon-pumped dye laser. Administering 86 J/cm2 total fluence (1 h irradiation) to Fisher rat thyroid cells during 2 h incubation with either daunomycin (excitation wavelength: 488 nm) or 5-iminodaunomycin (595 nm) produced cell killing at doses (about 2.7 X 10(-7) M for 50% cell survival) which were not toxic if administered in the dark. Greater photocytotoxicity (about 7 X 10(-8) M for 50% cell survival) was obtained with 4-demethoxydaunomycin as well as with its 6- and 11-amino derivatives (514 nm) while no cell killing as a result of photosensitization was observed for either Adriamycin or its 4'-iodo derivative. Our results suggest that the photosensitizing efficacy correlates with the absence of the methoxy group in the anthraquinone chromophore but is rather independent of the occurrence of triplet-mediated photoreactions. Finally, the fact that the imino- or amino-substituted 4-demethoxy compounds exhibit red-shifted absorption spectra compared to the parent molecule might be exploited for in vivo applications of the photoactivated cytotoxicity reported in this work.     

Influence of incubation time and sensitizer localization on meta-tetra(hydroxyphenyl)chlorin (mTHPC)-induced photoinactivation of cells

The present study addresses the impact of different aggregation states of meta-tetra(hydroxyphenyl)chlorin (mTHPC) on the photoinactivation of cells. Measurements of the photophysical properties of mTHPC in MCF-7 cells showed progressive sensitizer aggregation with increasing incubation time. Reconstructed absorption spectra of intracellular mTHPC showed a significant decrease in the molar extinction coefficient and broadening of the Soret band at 24 h incubation compared to 3 h. Intracellular photobleaching of mTHPC slowed down, and the profile changed from mono- to bi-exponential upon incubation. Fluorescence lifetime imaging (FLIM) measurements revealed a substantial decrease in the lifetime of mTHPC fluorescence at 24 h compared to 3 h. In addition, the intracellular localization of mTHPC as observed by fluorescence microscopy changed from a diffuse homogeneous fluorescence pattern at short incubation times to a punctiform pattern at 24 h. The efficiency of photodynamic therapy (PDT) assessed by a clonogenic assay was three times greater at 24 h. However, when the survival curves were replotted as a function of the number of absorbed photons, the efficiency was 1.8 times greater at 3 h than at 24 h. The loss of photosensitizing efficiency at higher mTHPC concentrations was attributed to self-quenching of the triplet states of the sensitizers.     

The metallochromic indicator dye Arsenazo III forms 1: 1 complexes with caffeine

The metallochromic indicator dye, Arsenazo III, forms a 1:1 complex with caffeine, a methylxanthine. Binding is accompanied by a wavelength-dependent shift in the absorption spectrum of the dye. The magnitude of the absorption change is significant at wavelengths typically used to monitor intracellular calcium ion. The equilibrium constant for the caffeine-dye reaction is approx. 20 mM. The complex has a differential molar extinction coefficient of ?5.05 · 10³ M^?1 · cm^?1 at 630 nm.     

Spectrophotometric titration of tyrosyl residues in alkaline mesentericopeptidase.

At pH 7.0 the alkaline mesentericopeptidase has ultraviolet absorption spectrum with a minimum at 251 nm and a maximum at 280 nm and no visible absorption. From the tyrosine to tryptophan ratio a value of 3 tryptophyl residues per mole of protein is obtained. The molar extinction coefficient at 280 nm is 3.55 X 10(4)M-1cm-1. Spectrophotometric titration studies show that the molecule of mesentericopeptidase contains seven phenolic groups with a pKapp - 9.92 and four to five groups with a pKapp = 11.96. Denaturing agents, such as 5 M guanidine hydrochloride or alkali, normalize the ionization of the tyrosyl residues. There is a good correlation between the spectrophotometric titration data and the results for the reactivities of the tyrosines in mesentericopeptidase towards tetranitromethane. The correlation is explained by the mechanism of nitration. Conclusions about the state of the tyrosyl residues and the three-dimensional structure of mesentericopeptidase are made.     

Romanowsky dyes and Romanowsky-Giemsa effect. 5. Structural investigations of the purple DNA-AB-EY dye complexes of Romanowsky-Giemsa staining.

A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations. Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18,100 cm-1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB approximately 1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions. The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly oriented dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solubility and diffusion coefficient of adenosine 3':5'-monophosphate.

Several previously unavailable parameters of adenosine 3':5'-monophosphate have been determined. The molar extinction coefficient at pH 7.0 is 1.38 X 10(-4), the aqueous solubility at pH 7.0 is 0.0236 M and the diffusion coefficient is 4.44 X 10(-6) cm2/s.