Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding

The quenching of fluorescence (up to 98%) by anti-fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti-fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F-(CH(2))(n)-T. These F-(CH(2))(n)-T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti-fluorescein antibody, however, up to 110-fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays.     

The ultrastructure of the uterine epithelium of the pig during the estrous cycle and early pregnancy

Summary In the compound eye of the moth Antheraea polyphemus, three types of visual pigments were found in extracts from the retina and by microspectrophotometry in situ. The absorption maxima of the receptor pigment P and the metarhodopsin M are at (1) P 520–530 nm, M 480–490 nm; (2) P 460–480 nm, M 530–540 nm; (3) P 330–340 nm, M 460–470 nm. Their localization was investigated by electron microscopy on eyes illuminated with different monochromatic lights. Within the tiered rhabdom, constituted of the rhabdomeres of nine visual cells, the basal cell contains a blue-and the six medial cells have a greenabsorbing pigment. The two distal cells of most ommatidia also have the blue pigment; only in the dorsal region of the eye, these cells contain a UV-absorbing pigment, which constitutes a portion of only 5% of the visual pigment content within the entire retina. The functional significance of this distribution is discussed.     

The Sonogashira reaction as a new method for the modification of borated analogues of the green fluorescence protein chromophore

The Sonogashira reaction was used for modifications of borated green fluorescence protein chromophore derivatives, 4-(2-(difluoroboryl)benzylidene)-1H-imidazol-5(4H)-ones, for the development of new fluorescent dyes. The derivatives bearing an acetylene fragment and a difluoroboryl group were obtained in high yields. The modification resulted in a significant bathochromic shift of the absorption and emission maxima and is a promising method for the development of new fluorescent dyes.     

一品红苞片花色素的分离及初步鉴定

用紫外-可见光分光光度计、高效液相色谱（HPLC）和质谱（MS）技术对一品红（Euphorbia pulcherrima）红色苞片中的花色素提取液进行了初步鉴定.一品红花色素的甲醇溶液分别在270、340和520 nm处有3个吸收峰;在440 nm吸光度与可见光最大吸收波长520 nm吸光度的比值为0.29;花色素的甲醇溶液中加入AlCl3后发生红移,再加入HCl后发生蓝移;色素溶液在紫外光下无荧光;色素样品经液相色谱分离后在270 nm检测有5个比较明显的吸收峰;质谱中得到595、611、381、571和589等对应的分子离子峰;花色素酸解液高效液相色谱图谱和鼠李糖、葡萄糖的出峰时间一致.由这些结果可推断一品红花色素样品中主要含有5种组分：矢车菊花色素芸香苷、飞燕草花色素芸香苷、飞燕草花色素苯甲酰基葡糖苷、矢车菊花色素苯甲酰基葡糖苷和一种未知成分.     

Fluorescent rhodol derivatives: versatile, photostable labels and tracers.

A series of chemically reactive, fluorescent rhodol derivatives was prepared and evaluated. Reactive functional groups included activated esters, amines, haloacetamides, fixable hydrazide derivatives, acrylamides, and photoaffinity reagents. Depending on the choice of substituents, absorption maxima of the dyes varied from 490 to 550 nm with extinction coefficients that were generally greater than 50,000 M-1 cm-1 in aqueous solution and emission maxima from 520 to 580 nm. Most of the compounds investigated exhibited fluorescence lifetimes between 3 and 4 ns. Individual derivatives were suitable for excitation with the 488 and 514-nm lines of the argon ion laser and the 546-nm line of the mercury arc lamp and were compatible for use with standard fluorescein and rhodamine filter sets. The rhodol dyes were more photostable and less sensitive to pH changes in the physiological range than fluorescein derivatives. Some examples show absorption maxima at or near 514 nm, an excitation wavelength that is useful for multicolor fluorescence microscopy, flow cytometry, and DNA sequencing. Derivatives were also prepared that exhibit absorption and emission maxima similar to those of tetramethylrhodamine (TMR) analogs but with higher quantum yields in aqueous solution. A number of the dyes had higher solubilities in aqueous systems and were less quenched on conjugation to proteins than TMR derivatives. Appropriate substitution results in a wider range of solubilities in hydrophilic or lipophilic solvents than is easily accomplished with fluorescein or TMR derivatives. Conjugates of a number of the rhodol fluorophores were generally more photostable and less pH sensitive than fluorescein conjugates and more fluorescent than TMR conjugates.     

一品红苞片花色素的分离及初步鉴定

用紫外-可见光分光光度计、高效液相色谱(HPLC)和质谱(MS)技术对一品红(Euphorbia pulcherrima)红色苞片中的花色素提取液进行了初步鉴定。一品红花色素的甲醇溶液分别在270、340 和520 nm 处有 3 个吸收峰; 在 440 nm 吸光度与可见光最大吸收波长 520 nm 吸光度的比值 为0.29; 花色素的甲醇溶液中加入AlCl3后发生红移, 再加入HCl后发生蓝移; 色素溶液在紫外光下无荧光; 色素样品经液相色谱分离后在 270 nm检测有 5 个比较明显的吸收峰; 质谱中得到 595、611、381、571 和 589 等对应的分子离子峰; 花色素酸解液高效液相色谱图谱和鼠李糖、葡萄糖的出峰时间一致。由这些结果可推断一品红花色素样品中主要含有 5 种组分: 矢车菊花色素芸香苷、飞燕草花色素芸香苷、飞燕草花色素苯甲酰基葡糖苷、矢车菊花色素苯甲酰基葡糖苷和一种未知成分。     

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c ₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t _1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t _1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center     

Acid denaturation of the B875 light-harvesting complex in membranes of Rhodobacter sphaeroides

The structural basis for the spectral red shift in the near-IR absorption band of the B875 light-harvesting complex was examined by treatment of membranes from Rhodobacter sphaeroides M21 with acid. This mutant strain lacks the overlapping spectral bands of the B800–850 light-harvesting antenna and gives rise to membrane fragments with both surfaces accessible to protons. At pH 2.2, about half the absorption at 876 nm was converted within 10 min to a free pigment band; the remaining absorption appeared at 880 nm and shifted to 845 nm over the next three hours. These spectral shifts could not be reversed by alkali. Approximately one-third of the characteristic near-IR CD signal of B875 was also lost initially and replaced by a broad trough centered near 854 nm. Thereafter, the CD spectrum was dominated by the strong conservative signal of the 845 nm absorbing component which was attributed to an oligomeric bacteriopheophytin a species, probably a dimer. A kinetic analysis of the acid-induced absorption changes suggested a multi-step model with rate constants of 0.37 min^-1 for the initial rapid change and 0.05 and 0.11 min^-1 for the respective subsequent steps. The non-conservative nature of the near-IR CD spectrum of the intact complex, together with the spectral changes observed after the initial loss of near-IR absorption and CD, suggest that pigment-pigment interactions are not solely responsible for the red shift in this complex.Abbreviations BChl bacteriochlorophyll a - BPheo bacteriopheophytin a     

Serotonin-immunoreactive and dopamine-immunoreactive neurones in the terminal ganglion of the cricket,Acheta domestica: Light- and electron-microscopic immunocytochemistry

Summary The distribution and ultrastructure of serotonin- and dopamine-immunoreactive (5-HTi and DAi) neurones have been investigated in the terminal ganglion of the cricket, Acheta domestica, using a pre-embedding chopper technique. Special attention has been paid to the immunoreactive structures in the neuropil. 5-HTi structures are extensively distributed and densely packed throughout the 5 neuromeres of the terminal ganglion and originate from several interneurones and efferent neurones. In contrast, DAi fibres are distributed sparsely although they extend to all neuromeres of the ganglion and originate from 6 interneurons only. For both 5-HTi and DAi neurones characteristic axonal projections and branching patterns can be distinguished. The 5-HTi axons exhibit rich varicose arborizations, whereas DAi neurones possess fewer varicosities in the neuropil. Electron microscopy shows that 5-HTi varicosities contain small ( 60 nm) and large ( 100 nm) agranular vesicles, and large ( 100 nm) granular vesicles, whereas in DAi varicosities small ( 60 nm) agranular and large ( 100 nm) granular vesicles are seen. Both 5-HTi and DAi varicosities form synaptic contacts. We conclude that both serotonin and dopamine may be used as neurotransmitters in the terminal ganglion of the cricket.Fellow of the Alexander von Humboldt-Stiftung     

The structure of cyanobacterial phycobilisomes: a model

Phycobilisomes, supramolecular complexes of water-soluble accessory pigments, serve as the major light-harvesting antennae in cyanobacteria and red algae. Regular arrays of these organelles are found on the surface of the thylakoid membranes of these organisms. In the present study, the hemi-discoidal phycobilisomes of several species of cyanobacteria were examined in thin sections of cells and by negative staining after isolation and fixation. Their fundamental structures were found to be the same. Isolated phycobilisomes possessed a triangular core assembled from three stacks of disc-shaped subunits. Each stack contained two discs which were 12 nm in diameter and 6–7 nm thick. Each of these discs was probably subdivided into halves 3–3.5 nm thick. Radiating from each of two sides of the triangular core were three rods 12 nm in diameter. Each rod consisted of stacks of 2 to 6 disc-shaped subunits 6 nm thick. These discs were subdivided into halves 3 nm thick.The average number of discs of 6 nm thickness forming the peripheral rods varied among the strains studied. For certain chromatically adapting strains, the average rod length was dependent upon the wavelength of light to which cells were exposed during growth. Analyses of phycobilisomes by spectroscopic techniques, polyacrylamide gel electrophoresis, and electron microscopy were compared. These analyses suggested that the triangular core was composed of allophycocyanin and that the peripheral rods contained phycocyanin and phycoerythrin (when present). A detailed model of the hemi-discoidal phycobilisome is proposed. This model can account for many aspects of phycobiliprotein assembly and energy transfer.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin - AP-B allophycocyanin B - C- cyanobacterial - R- rhodophytan - B- Bangiophycean - SDS sodium dodecyl sulfate - LPP Lyngbya-Plectonema-Phormidium group - Na-KPO₄ buffers NaH₂PO₄ titrated with a solution of KH₂PO₄ of equivalent molarity to a given pH     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

Regulation of the distribution of excitation energy in Ochromonas danica,an organism containing a chlorophyll-A/C/carotenoid light harvesting antenna

A chlorophyll a, c-fucoxanthin pigment-protein complex8 functions as the major light harvesting antenna in the Chrysophyte Ochromonas danica. The regulated distribution of excitation energy between the two photosystems was investigated in these organisms and was shown to be strongly wavelength dependent. A light state transition was induced by pre-illumination of cells using light 2 (640 nm) and light 1 (700 nm) of equal absorbed intensity, and detected by reversible changes in the 77 K chlorophyll fluorescence emission spectra. Peaks at 690 nm and 720 nm in the low temperature spectra are most likely associated with PS2 and PS1 respectively. A room temperature fluorescence emission at 680 nm induced by modulated light 2 (500 nm) was strongly quenched in the presence of background light 1 (720 nm). Removal of light 1 led to an increase in fluorescence followed by a slow quenching. The room temperature fluorescence changes were directly correlated with changes in the 77 K emission spectra that indicated a change in the distribution of excitation energy between the two photosystems. It was established that DCMU (1 mol) prevented the state 2. The conversion to state 1 followed a simple photochemical dose dependence and had a half-time of 20 s-1.5 min at 6 W m^-2. In contrast, the conversion to state 2 was independent of light intensity. These data indicate that O. danica undergoes a light state transition in response to the preferential excitation of PS2 or PS1.Abbreviations PS2 photosystem 2 - PS1 photosystem 1 - LHC light harvesting chlorophyll a/b protein - fx fucoxanthin - PQ plastoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea     

Membrane differentiation in the cytoplasmic-tubule system of the intestinal absorptive cells of the lamprey,Lampetra japonica

Summary Specific membrane differentiation occurs in the cytoplasmic-tubule system of the absorptive cells lining the mucosa of the lamprey anterior intestine. The absorptive cells are characterized by the presence of abundant mitochondria and a system of well-developed cytoplasmic tubules (120 nm in diameter). The cytoplasmic tubules open on to the basolateral cell surface and contain numerous lipoprotein particles (50–100 nm diam.) in their lumina. Lipoprotein particles are also observed in the endoplasmic reticulum and the Golgi complex, and they are transfered to the lateral intercellular space and lamina propria by way of the cytoplasmic tubules. Spirally-wound parallel rows of particles are found in the luminal surface of the cytoplasmic tubules. The rows are 17 nm apart and are wound spirally at a pitch of 210 nm. Freeze-fracture images of the tubule membranes also show spiral arrays of particles (9 nm in diameter) on the P-face, and complementary shallow grooves on the E-face. From these observations, it is suggested that the cytoplasmic-tubule system of the intestinal absorptive cells serves as a channel for the transport of synthesized lipoprotein into the interstitium, and is also the site of the ion and water exchange essential for the maintenance of ionic homeostasis.     

Time resolved fluorescence of bacteriophage Pfl DNA binding protein and its complex with DNA

The DNA binding protein of the filamentous bacteriophage Pfl exhibits fluorescence from a single tryptophan residue. The location of the emission maximum at 340 nm ist quite common for proteins, but the single lifetime of 7.8 ns is one of the longest yet reported. Protein fluorescence is quenched more efficiently by Cs⁺ than by I^-; the Trp is located in a partially exposed pocket, in the vicinity of a negative charge.In the native complex of the binding protein with Pfl DNA the fluorescence emission maximum is at 330 nm, indicating a more apolar environment for Trp 14. The native nucleoprotein complex exhibits a similar fluorescence lifetime (6.5 ns) and an approximately equal fluorescence yield, indicating the absence of Trp-DNA stacking. The tryptophan in the complex is virtually inaccessible to ionic quenchers, and thus appears to be buried.Fluorescence depolarisation measurements have been used to examine the rotational mobility of the tryptophan in the protein and in the nucleoprotein complex. In the protein alone a single rotational correlation time () of 19 ns is observed, corresponding to rotation of the entire dimeric molecule; in the native nucleoprotein complex with Pfl DNA, a of 500 ns is observed, corresponding to a rigid unit of at least 50 subunits. In neither case does the tryptophan exhibit any detectable flexibility on the subnanosecond time scale.     

Synthesis and Properties of Bacteriorhodopsin Analogs Containing Electron-Density Labels in the Chromophore Moiety

A method for synthesis of retinal analogs labeled with electron-density groups is suggested. The interaction of these polyene compounds with bacterioopsin in apomembrane of Halobacterium salinarum was tested. A retinal analog containing a crown-ether receptor group is able to interact readily with bacterioopsin giving rise to rapid formation of a pigment with absorption maximum at 460 nm. This pigment is capable of undergoing cyclic photoconversion. The crown-bacteriorhodopsin photocycle is extremely slow and its quantum efficiency is very low (3% of that in native bacteriorhodopsin). This photocycle includes an M-like intermediate with a differential absorption maximum at 380 nm. A retinal analog in which the -ionone ring is replaced by ferrocene moiety forms a stable chromoprotein with the main absorption band at 483 nm and a shoulder near 590-610 nm.     

Preparation of fragments 505-582 and 505-573 of bovine serum albumin

Peptic fragment 505-582 of bovine serum albumin, the "Phe" fragment, has been found useful in several laboratories for studies of antigenic sites, ligand binding and metabolism. It contains the entire carboxyterminal loop, Loop 9, of the albumin molecule. We present an improved preparation of this peptide. Peptide 505-582 is obtained in about 9% yield, along with a similar yield of the closely associated peptide 505-573 arising from a second peptic cleavage. The unusual ultraviolet absorption spectrum of these peptides shows triple maxima near 259 nm reflective of the high phenylalanine content and the total absence of tyrosine and tryptophan.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

Comparative photophysical investigation of oxygen and sulfur as covalent linkers on octaalkylamino substituted zinc(II) phthalocyanines.

This work describes a systematic comparison of oxygen and sulfur as covalent linkers on octasubstituted zinc(II) phthalocyaninates. Most photophysical parameters that make phthalocyanines technologically relevant, e.g. molar absorption coefficients, fluorescence, triplet and singlet oxygen quantum yields, are essentially unaffected by the substitution. The energy content of the first triplet state was observed to be close to the first singlet state of molecular oxygen for both spacers, as follows from photoacoustic determinations. Nonetheless, a bathochromic shift of 30 nm in the absorption and emission maxima, and of 60 nm in the triplet-triplet absorption spectra were observed when alkyloxyl and alkylsulfanyl moieties were alternatively present. Fluorescence quantum yields proved to be much more sensitive towards aggregation than the absorption spectra. Therefore, a novel fluorescence data analysis provided aggregation parameters and photophysical properties of the monomeric species. It was observed that the tendency towards dimerization is slightly higher with sulfur linkers. These results set a foundation for the rational design of conveniently substituted phthalocyaninates with different connectors between the macrocycle and the side chains.     

Mikrospektralphotometrische Untersuchungen zum Speichermechanismus des amphoteren OxazinfarbstoffsPrune pure durch die lebende pflanzliche Zelle

Zusammenfassung Elektrophorese-, spektralphotometrische und Ausschüttelversuche mit hydrophoben Solventien ergeben, daßPrune pure in der wässerigen Lösung, je nach dem pH-Wert, als einwertiges Kation, Zwitterion und Anion vorliegt. Im stark sauren Bereich (H₂SO₄) kann es noch als zweiwertiges Kation auftreten. Das Farbbasenmolekül ist nur in hydrophoben Lösungsmitteln nachzuweisen.Mit Zunahme der Farbstoffkonzentration in einer wässerigen Lösung (pH7) verschiebt sich das Absorptionsmaximum von = 640 nm nach = 580 nm. Diese Verschiebung beruht sehr wahrscheinlich auf einer Assoziation der Zwitterionen.In polaren Lösungsmitteln verschiebt sich das Absorptionsmaximum desPrune pure mit zunehmender Polarität in den längerwelligen Bereich.Rutin bedingt eine negative Metachromasie.Plasma und Zellkern der Oberepidermis vonAllium- cepa- Schuppenblättern zeigen nach Vitalfärbung mitPrune pure übereinstimmend ein breites Absorptionsmaximum bei 575 nm. Die Färbungsintensität übt keinen Einfluß auf die Lage des Maximums aus.Die mitPrune pure gefärbten vollen Zellsäfte der Unterepidermis besitzen ein Absorptions-maximum bei 675 nm.Aus der Lage der Absorptionsmaxima und der Form der Absorptionskurven wird geschlossen, daßPrune pure von Plasma und Zellkern als Farbbasenmolekül in apolaren Lipoiden gespeichert wird. Auf Grund der Dissoziationskonstante des Farbstoffs kannPrune pure nur von vollen Zellsäften aufgenommen werden. In den Unterepidermiszellen liegt eine Bindung an zellsafteigene Flavonole vor.

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Microspectrophotometric investigations into the mechanism of accumulation of the amphoteric oxacin dyePrune pure by the living plant cell

Summary Electrophoretic, spectrophotometric and shake out experiments withPrune pure show that in aqueous solution the dye is present according to pH as a univalent cation, zwitterion or anion. In strongly acid solutions (H₂SO₄) it may be present, too, as a bivalent cation. The dye base molecule is demonstrable only in hydrophobic solvents.In aqueous solution (pH 7.0) the absorption maximum shifts from = 640 nm to = 580 nm when the concentration of the dye is increased. The shift probably is due to an association of zwitterions.In polar solvents the absorption maximum ofPrune pure shifts towards the long wave area with increasing polarity of the solvent.Rutin causes negative metachromasy.After vital staining withPrune pure both the plasma and the nucleus of cells from the upper epidermis of scale leaves ofAllium cepa show a broad absorption maximum at 575 nm. The staining intensity has no influence over the position of the maximum.The full cell saps of cells from the lower epidermis give an absorption maximum at 675 nm when stained withPrune pure.From the position of the absorption maxima and the shape of the absorption curves it is concluded thatPrune pure is accumulated by the plasma and the nucleus as the dye base molecule into apolar lipoids.According to the dissociation constant ofPrune pure the dye can only be accumulated by ful cell saps. In cells of the lower epidermis it is bound to flavonoles present in the cell sap.

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Herrn Professor Dr. H.Drawert danke ich für die Unterstützung dieser Arbeit und für anregende Diskussionen; dem Deutschen Akademischen Austauschdienst (DAAD) bin ich für die Gewährung eines Stipendiums dankbar. Das UMSP I stellte die Deutsche Forschungsgemeinschaft zur Verfügung.     

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.