Em Max Research Articles

A key role for the mitochondrial benzodiazepine receptor in cellular photosensitisation with δ-aminolaevulinic acid

The aim of this study was to determine the part played by the mitochondrial benzodiazepine receptor in cellular photosensitisation with the protoporphyrin IX precursor, δ-aminolaevulinic acid. Evaluation of the δ-aminolaevulinic acid-concentration dependence and kinetics of fluorescent protoporphyrin IX accumulation in monolayers of rat AR4 2J pancreatoma cells established a basis for assessing pharmacological modulation of the biosynthetic pathways for protoporphyrin IX production and photocytotoxicity. Iron chelation enhanced the accumulation of photo-active protoporphyrin IX whereas 1-(2-chlorophenyl)- N-methyl- N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), dipyridamole, or 7-(dimethylcarbamoloxy)-6-phenylpyrrolo-[2,1- d]benzothiazepine (DPB), competitive ligands of the mitochondrial benzodiazepine receptor, all diminished protoporphyrin IX accumulation, as did acifluorfen, a mitochondrial protoporphyrinogen oxidase inhibitor. In addition to protoporphyrin IX (Em max: 630 nm), δ-aminolaevulinic acid-treated cells also generated a fluorophore of Em max 580 nm; this compound was identified as Zn-protoporphyrin IX. Mitochondrial benzodiazepine receptor ligands increased the formation of the zinc porphyrin whilst decreasing that of protoporphyrin IX. The involvement of the mitochondrial benzodiazepine receptor in the translocation of porphyrins and the formation of Zn-protoporphyrin IX have wide implications for the use of δ-aminolaevulinic acid in photodynamic therapy.

Characterization of the fluorescence emission properties of prodan in different reverse micellar environments

We have examined the steady state and time resolved fluorescence emission properties of the hydrophobic fluorescence probe, prodan, in three representative reverse micellar systems formed by the surfactants poly(oxyethylene) (tetramethylbutyl) phenylether (Triton X-100, neutral), cetyl trimethylammonium bromide (CTAB, cationic) and sodium bis-(2-ethylhexyl) sulfosuccinate (AOT, anionic) in organic solvent media containing different concentrations of water. The results obtained from the experiments indicate conspicuous dependence of the emission behaviour of prodan on the type of surfactant used and the water/surfactant molar ratio ( w 0). The nature of the emission profiles, along with relevant parameters namely emission maximum ( λ em max), anisotropy ( r) and lifetime ( τ) data are used to infer the distribution and microenvironments of the prodan molecules in the reverse micelles at different w 0 values. Furthermore, quantitative estimates have been obtained for the polarities (in terms of the empirical polarity parameter E T(30)) of the sites of solubilization of the fluorophore in different reverse micellar systems.

Luminescence behaviour of 5-hydroxyindole in different environments

Steady state fluorescence emission spectroscopic studies along with some lifetime measurements have been performed for 5-hydroxyindole (5HI) in different environments. 5HI merits particular attention, since it is the chromophoric moiety of the non-natural amino acid 5-hydroxytryptophan (5HT), which has come into significant recent prominence as a novel intrinsic optical probe for protein structure, function and dynamics. Studies in representative homogeneous solvents and solvent-mixtures indicate that unlike other fluorophores of related interest like indole (I) and 7-azaindole (7AI), the fluorescence emission maximum ( λ em max) of 5HI is relatively insensitive to solvent polarity. This behaviour suggests the lack of appreciable solvent dipolar relaxation in 5HI, which is consistent with our low temperature (77 K) emission data. Notwithstanding such limitation, fluorescence anisotropy ( r) and quenching studies are shown to be effective for exploring changes in the micro-environments of 5HI in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT) reverse micellar assemblies (which serve as a biomembrane mimetic model system) with variation in water/surfactant molar ratio ( w 0).

Three coordinate copper (I) complexes. Synthesis, spectral, and structural study of copper (I) complexes of 4-benzoylpyridine and X-ray crystal structure of the dimer [CuCl (4-benzoylpyridine) ] 2

Yellow to red–brown copper (I) complexes of the type CuXL for X=Cl, Br,I, CN, SCN and N 3 and CuXL 2 for X−NO 3 and ClO 4, where L=4-benzoylpyridine, have been synthesized and characterized. All complexes exhibit MLCT bands in the visible region and the neutral complexes gave non-conducting solutions in DMF. The IR and Raman spectral data suggest monodentate 4-Bzpy, bridging halides or pseudohalides, bidentate nitrate and perchlorate in the respective complexes in the solid state. The structure of the 1:1 chloride complex, as determined by X-ray crystallography, features a discrete centrosymmetric [CuCl (4-benzoylpyidine) ] 2 molecule with a Cu···Cu distance of 2.953 Å. Each copper atom in the dimeric molecule is trigonally planar coordinated by two μ-chlorine atoms and the nitrogen atom of 4-benzoylpyridine. All the isolated complexes display emission spectra in the solid state at room temperature. The neutral halide or pseudohalide complexes show one emission band with λ em max in the range 586–642 nm which is independent of the excitation wavelength and is related to MLCT or XLCT excited state. The 1:2 nitrate and perchlorate complexes exhibit their λ em max around 468–504 nm.

An assessment of the usefulness of 5-hydroxytryptophan as an optical probe

5-Hydroxytryptophan (5HT) has been the focus of much recent attention as a novel intrinsic fluorescence probe for proteins. Solvent-dependence studies reported here show that, compared to other fluorophores of related interest like tryptophan (Trp) and 7-azatryptophan (7AT), the fluorescence emission maximum ( λ em max) of 5HT is relatively insensitive to solvent polarity. Thus, upon change of solvent from acetonitrile to water, the λ em max of 5HT fluorescence is red shifted by only 3 nm, in contrast to corresponding red shifts of much larger magnitudes observed for Trp and 7AT (14 and 23 nm, respectively). This behaviour suggests the lack of significant solvent dipolar relaxation effects in 5HT, which is corroborated by the low temperature emission measurements at 77°K. This aspect is also revealed in the fluorescence characteristics of 5HT in a membrane-mimetic model system, namely in AOT reverse-micellar assemblies containing different amounts of water. With increase in water content, the λ em max of 5HT remains almost unaffected, although fluorescence anisotropy and quenching data clearly indicate significant changes in the microenvironments of 5HT molecules.

Effect of mutations at Cys 237 on the activation state and activity of human phenylalanine hydroxylase

Wild-type recombinant human phenylalanine hydroxylase (wt-hPAH) is activated about 1.5-fold by exposure to alkaline pH (pH 8.5–9.0). In order to study whereas this activation might be related to the activation of the rat enzyme by N-ethylamaleimide-modification of Cys 237 [Gibbs and Benkovic (1991) Biochemistry 30, 6795], mutant proteins of hPAH with Cys 237 changed to Ser (S) or Glu (D) have been prepared. The mutant forms have high specific activity at pH 7.0 and high affinity for l-Phe, notably for hPAH-C237D, which shows a 3-fold higher activity than l-Phe-activated wt-hPAH and it is not further activated by pre-incubation with l-Phe. Moreover, the emission maximum of the intrinsic fluorescence of hPAH-C237D (λ max em=347 nm) resembles that of activated forms of wt-hPAH. However, the activity of this mutant at neutral pH is further activated by exposure to alkaline pH, indicating that activation of wt-hPAH by alkaline pH is not restricted to ionization of Cys 237.

Probing the cysteine 34 residue in human serum albumin using fluorescence techniques

The microenvironment surrounding Cys-34 in human serum albumin (HSA) has been studied using acrylodan, a cysteine-specific fluorescence probe. Fluorescence emission maximum (Em max) of acrylodan covalently attached to Cys-34 was observed at 476 nm, which was nearly the same as for acetonitrile. The Em max exhibited a shift toward longer wavelength with oleate binding. The acrylodan fluorescence of HSA exhibited heterogeneous decay kinetics, which adequately fit a double-exponential decay model. When three or more oleate molecules were bound to one albumin molecule, the fractional intensity was shifted in favor of the shorter lived component. These results suggest that oleate binding induces a conformational or dynamic change which is localized in the Cys-34 region. Unfolding studies with guanidine-HCl indicate that Cys-34 appears to be located on the surface of HSA molecule and that it is protected by adjacent amino acid residues. Solvent accessibility of acrylodan with HSA in the absence and presence of oleate was determined from acrylamide quenching, and suggests that oleate binding enhances the solvent exposure of the acrylodan fluorophore. In order to determine the nature of the electrostatic potential near Cys-34, the quenching rate constants for anionic (iodide) and cationic (thallium) quenchers were determined as a function of ionic strength of solvent. The ionic strength dependence of quenching indicated that there was an electrostatic attractive force between the fluorophore and both ionic quenchers. These results are consistent with a model in which amphoteric charges which arise from charged amino acid residues were surrounding Cys-34. Interestingly, oleate binding resulted in changes in the spatial relationships between acrylodan and these charged residues. Thus, the experiments described herein provide the information concerning an oleate-induced alteration in the nature of the local environment surrounding Cys-34 and suggests that long chain fatty acid binding provides a method for regulating the radical-trapping antioxidant activity of Cys-34 in HSA in vivo.

Premicellar aggregates in a mixed system of a surfactant (SDS) and polymer (EHEC)

Using steady-state fluorescence spectroscopy, the interaction of a non-ionic polymer, ethyl (hydroxyethyl) cellulose (EHEC) with premicellar aggregates of the anionic surfactant SDS formed below the critical micelle concentration (c.m.c.) was studied through the incorporation of a fluorescent probe, benzyl 1-pyrenoate (1-PirBz) into these aggregates. The monomer emission of 1-PirBz ( λ em max=410 nm, I m), which is the only emissive species found above the c.m.c., is accompanied by a broad structureless emission band ( λ em max=530 nm, I agg) possibly formed from a ground-state dimer within the premicellar aggregate. The monomer emission is strongly dependent on the micropolarity, microviscosity and ionic strength in the probe vicinity. The SDS concentration determines the formation of aggregates where concentrations increase up to 1 × 10 −3 M where a maximum ratio I agg: I m is obtained. Above this concentration the nature of premicellar aggregates is rather similar to that of micelles and the dimer formation decreases. Addition of polymer (EHEC) up to 1% to aqueous solutions containing SDS in the concentration range 0.1–3 mM leads to polymer-associated premicellar aggregates where two similar emissions appear. However, the monomer one shows some vibrational resolution measured by I m ( λ em=391 nm, 410 nm) and the dimer is shifted to the blue I agg ( λ em=518 nm). Below the critical aggregation concentration (c.a.c.) the polymer interacts with surfactant molecules, forming less-polar mixed premicellar aggregates, which can, however, coexist with polymer-free premicellar aggregates above a “polymer saturation concentration” by surfactant. While the surfactant c.m.c. is not significantly affected by temperature in the absence of polymer, the surfactant c.a.c. in the presence of 1% polymer (EHEC) is found to decrease as the temperature is increased.

Characterization of marine and terrestrial DOM in seawater using excitation-emission matrix spectroscopy

Abstract High-resolution fluorescence spectroscopy was used to characterize dissolved organic matter (DOM) in concentrated and unconcentrated water samples from a wide variety of freshwater, coastal and marine environments. Several types of fluorescent signals were observed, including humic-like, tyrosine-like, and tryptophan-like. Humic-like fluorescence consisted of two peaks, one stimulated by UV excitation (peak A) and one by visible excitation (peak C). For all samples, the positions of both excitation and emission maxima for peak C were dependent upon wavelength of observation, with a shift towards longer wavelength emission maximum at longer excitation wavelength and longer wavelength excitation maximum at longer emission wavelength. A trend was observed in the position of wavelength-independent maximum fluorescence ( Ex max Em max ) for peak C, with maximum at shorter excitation and emission wavelengths for marine samples than for freshwater samples. Mean positions of these maxima were: rivers Ex max Em max = 340 448 nm; coastal water Ex max Em max = 342 442 nm; marine shallow transitional Ex max Em max = 310 423 nm; marine shallow eutrophic Ex max Em max = 299 389 nm; and marine deep Ex max Em max = 340 438 nm. Differences suggest that the humic material in marine surface waters is chemically different from humic material in the other environments sampled. These results explain previous conflicting reports regarding fluorescence properties of DOM from natural waters and also provide a means of distinguishing between water mass sources in the ocean.

Use of 2,3-naphthalenedicarboxaldehyde derivatization for single-cell analysis of glutathione by capillary electrophoresis and histochemical localization by fluorescence microscopy.

We report that 2,3-naphthalenedicarboxaldehyde reacts rapidly with glutathione and its precursor, gamma-glutamylcysteine, to form highly fluorescent derivatives under physiological conditions. In contrast to previous accounts of 2,3-naphthalenedicarboxaldehyde labeling of primary amines, no additional CN- ion or any other additional nucleophile is required. The fluorescence spectral properties of the chromophores (lambda exc max = 472 nm, lambda em max = 528 nm) make these derivatives amenable to excitation and detection by optical instrumentation that is optimized for fluorescein wavelengths. This selective labeling chemistry enabled quantitative determination and histochemical localization of glutathione in neurobiological samples. Intracellular glutathione was labeled by incubating cultured cells or cell suspensions in a 2,3-naphthalenedicarboxaldehyde-supplemented, DMSO-containing physiological buffer (pH = 7.4) for 2-10 min. Applications include imaging of cultured NG 108-15 cells (mouse neuroblastoma x rat glioma) and primary glial and neuronal cell cocultures (rat hippocampus) using epiluminescent and confocal fluorescence microscopy. Quantitative determination of glutathione in single NG 108-15 cells was accomplished using laser-induced fluorescence detection and capillary electrophoresis.

Fluorescence properties of methyl 8-(2-anthroyl) octanoate, a solvatochromic lipophilic probe

Fluorescence excitation and emission spectra, relative fluorescence quantum yield φ r and fluorescence lifetime τ of methyl 8-(2-anthroyl)-octanoate have been studied in a set of organic solvents covering a large scale of polarity and in the presence of water. In this probe, the 2-anthroyl chromophore exhibits quite remarkable and unique fluorescence properties. Thus, when going from n-hexane to methanol, the maximum emission wavelength λ em max shifts from 404 nm to 492 nm while φ r and τ increase from 1 to 17.7 and from 0.91 ns to 13.5 ns, respectively. These increments are still more accentuated in the presence of water with estimated values of 526 nm for λ em max, 27 for φ r and 20 ns for τ in this solvent. Because of the presence of a keto group which is a hydrogen bond acceptor and which can conjugate with the aromatic ring so as to provide the chromophore with a high dipole moment, the fluorescence properties of the probe strongly depend on the polarity of the surrounding medium. They can be accounted for in terms of general solvent effects (dipolar solute/solvent interactions) in the presence of aprotic solvents and in terms of specific solvent effects (hydrogen bonding) in protic solvents. Such properties of solvatochromism make the 2-anthroyl chromophore, after 8-(2-anthroyl)octanoic acid has been attached to phospholipids (E. Perochon and J.F. Tocanne (1991) Chem. Phys. Lipids 58, 7–17) a potential tool for studying microenvironmental polarity in biological membranes.

Laser photobleaching leads to a fluorescence grade adenosine deaminase

The enzyme adenosine deaminase (adenosine amino-hydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at λ max ex = 350 nm and λ max em = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence ( λ max ex = 305 nm, λ max em = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

Reaction of Malonaldehyde with Nucleic Acid. II. Formation of Fluorescent Pyrimido[1,2-a]purin-10(3H)-one Mononucleotide

Abstract The reaction of malonaldehyde under acidic conditions (pH 4.5) with guanosine 5′-monophosphate resulted in the formation of fluorescent 3-(β-d-ribofuranosyl)pyrimido[1,2-a]purin-10(3H)-one 5′-phosphate (3a). This adduct was also isolated from malonaldehyde-reacted RNA. The amount of 3a in the modified RNA was estimated to be 0.4 per cent by weight. The fluorescence spectrum of 3a (Ex max. 360 nm, Em max. 500 nm) was similar to that of guanine- and guanosine-malonaldehyde adducts which have the same type base structure. On the other hand, another type of fluorophore (Ex max. 390 nm, Em max. 460 nm) was also formed in the reaction of malonaldehyde with a nucleic acid polymer. Thus, at least two different types of fluorophores are present in malonaldehyde-reacted nucleic acid.

A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibrolasts

A method for measuring the activity of the pyruvate dehydrogenase complex (PDC) by coupling acetyl-CoA production to acetylation of a fluorescent dye is described. Acetylation of cresyl violet acetate by pigeon liver acetyltransferase results in a shift of its fluorescence spectrum from λ ex max = 575, λ em max = 620 nm to λ ex max = 475, λ em max = 575 nm. The rate of appearance of acetylated dye was followed fluorometrically and was proportional to PDC activity in extracts of cultured human fibroblasts. The assay showed appropriate substrate and cofactor dependence and had a working range between 0.04 and 70 munits. It is 10 times more sensitive than the spectrophotometric assay on which it is based (working range 0.4–31 munits) and is equally convenient. Unactivated PDC activity in fibroblast extracts was 0.75 (0.60–0.92) munits/mg protein (mean and range for six cell lines).

Reaction of the Z isomer of 4-trans-(N,N-dimethylamino)cinnamaldoxime with the liver alcohol dehydrogenase-oxidized nicotinamide adenine dinucleotide complex.

The Z isomer of 4-trans-(N,N-dimethylamino)-cinnamaldoxime, (Z)-DMOX (lambda maxH2O 354 nm), forms a ternary complex with NAD+ and equine liver alcohol dehydrogenase. The 3-acetyl (3-acetyl-PdAD+), 3-thiocarboxamide (3-thio-NAD+), 3-iodo (io3PdAD+) and nicotinamide mononucleotide (NMN+) analogues of NAD+ also form ternary complexes with enzyme and (Z)-DMOX. These complexes are characterized by large red-shifts in the UV-visible spectrum of bound (Z)-DMOX (lambda max 428 nm for the NAD+ complex) and new spectral bands in the 280-340-nm region associated with the pyridine moieties of NAD+ and the NAD+ analogues. The ternary enzyme-NAD+-(Z)-DMOX complex is weakly fluorescent (lambda ex 430 nm; lambda em max 505 nm) and strongly quenches the residual tryptophan fluorescence of the enzyme-NAD+ binary complex. (Z)-DMOX binds with high affinity to the enzyme-NAD+ complex (Kd less than or equal to 4 X 10(-9) M at pH 8.75 and 25 degrees C), and similarly high affinities were found for the 3-acetyl-PdAD+, 3-thio-NAD+, and io3PdAD+ complexes. Binding is much weaker to the enzyme-NMN+ complex. The active site specifically substituted Co(II), Ni(II), Cu(II), and Cd(II) enzyme derivatives and the enzyme species lacking any metal ion at the active site (apoenzyme) also form ternary complexes with (Z)-DMOX in which the DMOX UV-visible spectrum is red-shifted (ranging from 43 to 83.5 nm). The complexes formed with the Zn(II) and Co(II) enzymes are characterized by relatively high affinities for (Z)-DMOX and by spectra that are independent of pH over the range 6-10. The affinity of the apoenzyme-NAD+ complex for (Z)-DMOX is much lower, and the spectrum of the complex is pH dependent with lambda max = 430 nm at pH 7 and lambda max = 397 nm at pH 10. The rate of (Z)-DMOX dissociation from the apoenzyme complex was found to be approximately 10(3)-fold greater than the rates observed for the metal ion substituted enzymes. The 280-340-nm spectral bands appear to result from the dihydropyridine moieties of covalent adducts formed between (Z)-DMOX and NAD+ and the NAD+ analogues. The large red-shifts of the (Z)-DMOX spectrum result from the bonding of the oxime nitrogen to a strong electrophilic center (either the active site zinc ion or the nicotinamide ring of NAD+.)(ABSTRACT TRUNCATED AT 400 WORDS)

Spatial relationship between a fast-reacting thiol and a reactive lysine residue of myosin subfragment 1.

Fluorescence energy transfer was used to examine the spatial proximity between two key side chains in myosin subfragment 1 (S-1), viz., the reactive thiol (SH1) located on the C-terminal 20K tryptic fragment and the reactive lysyl (RLR) on the N-terminal 27K tryptic fragment of S-1 heavy chain. S-1 was specifically labeled at SH1 with an energy donor, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS), and at RLR with an energy acceptor, 2,4,6,-trinitrobenzenesulfonate (TNBS). Prior blocking of SH1 with AEDANS increased the pK of RLR from 9.04 to 9.42. Trinitrophenylation of SH1-blocked S-1 was about 50% slower and sharply reduced the Ca2+ ATPase activity. Reciprocally, blocking of RLR with TNBS slowed the rate of reaction of SH1 and AEDANS by 40-60%. Addition of the second label does not grossly alter the conformation resulting from the first label. S-1 labeled at RLR with TNBS and at SH1 with optically inert iodoacetamide shows the same TNP difference spectrum +/- MgADP (lambda min 365 nm) as S-1 with S 1 free. Also, S-1 labeled at SH1 with AEDANS and at RLR with an optically inert methyl group shows the same AEDANS emission spectrum (lambda em max 475 nm), excited-state lifetime (tau = 20.3 ns) and rotational correlation time (phi = 106 ns) as S-1 with RLR free. When the decrease of either the quantum yield or the excited-state lifetime of the donor in the absence and presence of the acceptor was measured, the energy transfer efficiency was found to be 70%. The apparent interchromophore distance was calculated to be 2.6 nm through the use of the Förster equation with an uncertainty of less than 12%.

Reversed- and normal-phase high-performance liquid chromatography of gibberellin methoxycoumaryl esters

The methoxycoumaryl esters of thirteen gibberellins were synthesized by crown ether catalysis. These derivatives are highly fluorescent (λ ex max 320 nm, λ em max 400 nm) and can be detected at the low picogram level with a spectrophotofluorimeter after reversed-phase high-performance liquid chromatography. The various gibberellin esters were readily resolved by high-performance liquid chromatography on both ODS-Hypersil and CPS-Hypersil supports. There were marked differences in the behaviour of mono, bis and tris esters. Chemical ionization negative-ion mass spectra of the gibberellin methoxycoumaryl esters were obtained by direct-probe mass spectrometry.