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.     

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.     

Inhibition of lactate and malate dehydrogenase by dyes containing vinylsulfone and chlorotriazine groups

The difference spectra of lactate and malate dehydrogenase complexes with four native dyes containing vinylsulfonic and triazinic groups (light-resistant yellow 2KT, red-violet 2KT, etc.) were monitored in 0.1 M phosphate buffer pH 8.2 at 20 degrees C. The dissociation constants were calculated from the spectral data. The most stable complexes were lactate dehydrogenase--light-resistant yellow 2KT and malate dehydrogenase--light-resistant yellow 2KT ones. The values of delta H degree = 5.75 kcal/mole and standard thermodynamic parameters, delta G degree = -6.5 kcal/mole and delta S degree = 41.2 e. u., were calculated from the values of association constants for temperature dependence. The thermodynamic characteristics confirmed the key role of hydrophobic interactions in lactate dehydrogenase--reactive dye complex formation. All the dyes under study competitively inhibit lactate and malate oxidation by the corresponding dehydrogenases. The inhibition constants of both enzymes by the four dyes were determined at 20 degrees C in 0.1 M phosphate buffer pH 8.2. Light-resistant yellow 2KT appeared to be the most effective inhibitor of the enzymes.     

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)     

Thermal fluctuations between conformational substates of the Fe(2+)-HisF8 linkage in deoxymyoglobin probed by the Raman active Fe-N epsilon (HisF8) stretching vibration.

We have measured the VFe-His Raman band of horse heart deoxymyoglobin dissolved in an aqueous solution as a function of temperature between 10 and 300 K. The minimal model to which these data can be fitted in a statistically significant and physically meaningful way comprises four different Lorentzian bands with frequencies at 197, 209, 218, and 226 cm-1, and a Gaussian band at 240 cm-1, which exhibit halfwidths between 10 and 12.5 cm-1. All these parameters were assumed to be independent of temperature. The temperature dependence of the apparent total band shape's frequency is attributed to an intensity redistribution of the subbands at omega 1 = 209 cm-1, omega 2 = 218 cm-1, and omega 3 = 226 cm-1, which are assigned to Fe-N epsilon (HisF8) stretching modes in different conformational substrates of the Fe-HisF8 linkage. They comprise different out-of-plane displacements of the heme iron. The two remaining bands at 197 and 240 cm-1 result from porphyrin modes. Their intensity ratio is nearly temperature independent. The intensity ratio I3/I2 of the vFe-His subbands exhibits a van't Hoff behavior between 150 and 300 K, bending over in a region between 150 and 80 K, and remains constant between 80 and 10 K, whereas I2/I1 shows a maximum at 170 K and approaches a constant value at 80 K. These data can be fitted by a modified van't Hoff expression, which accounts for the freezing into a non-equilibrium distribution of substates below a distinct temperature Tf and also for the linear temperature dependence of the specific heat of proteins. The latter leads to a temperature dependence of the entropic and enthalpic differences between conformational substates. The fits to the intensity ratios of the vFe-His subbands yield a freezing temperature of Tf = 117 K and a transition region of delta T = 55 K. In comparison we have utilized the above thermodynamic model to reanalyze earlier data on the temperature dependence of the ratio Ao/A1 of two subbands underlying the infrared absorption band of the CO stretching vibration in CO-ligated myoglobin (A. Ansari, J. Berendzen, D. Braunstein, B. R. Cowen, H. Frauenfelder, M. K. Kong, I. E. T. Iben, J. Johnson, P. Ormos, T. B. Sauke, R. Scholl, A. Schulte, P. J. Steinbach, R. D. Vittitow, and R. D. Young, 1987, Biophys. Chem. 26:237-335).(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluorescence anisotropy studies of enzyme-substrate complex formation in stearoyl-ACP desaturase

Stearoyl-acyl carrier protein Delta(9)-desaturase (delta9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e(-) reduced delta9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting delta9D was observed with K(D1) = 13 +/- 3 nM. No significant difference in the K(D1) value was observed for 4e(-) delta9D. An approximately 4-fold increase in K(D1) per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting delta9D was estimated to have K(D2) approximately 350 +/- 40 nM. The K(D2) values exhibited a similar dependence on acyl chain length as observed for the K(D1) values. The k(off) values measured by stopped-flow anisotropy measurements for reversal of the enzyme-substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s(-)(1)) relative to 18:0-ACP (1 s(-)(1)). Increases in acyl chain length are thus associated with the presently reported increases in the K(D) and k(off) values. These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme-substrate complex between substrate release and subsequent steps in catalysis.     

The magnetic and electronic properties of Methanobacterium thermoautotrophicum (strain delta H) methyl coenzyme M reductase and its nickel tetrapyrrole cofactor F430. A low temperature magnetic circular dichroism study

Variable temperature magnetic circular dichroism (MCD) spectroscopy has been used to characterize the magnetic and electronic properties of the Ni(II) tetrapyrrole, F430, which is the cofactor of the S-methyl coenzyme M methylreductase enzyme from Methanobacterium thermoautotrophicum (strain delta H). 4-Coordinate forms are found to be diamagnetic (S = 0 ground state), whereas 6-coordinate forms are paramagnetic (S = 1 ground state). MCD studies, together with parallel low temperature UV-visible absorption and resonance Raman investigations, show that the equilibrium distribution of 4-coordinate square-planar and 6-coordinate bis-aquo forms of the native isomer of F430 in aqueous solution is affected by both temperature and the presence of glycerol. In the presence of 50% glycerol, the 12,13-diepimer of F430 is shown to be partially 6-coordinate in frozen solution at low temperature. Low temperature MCD magnetization data allow the determination of the axial zero-field splitting (D) of the S = 1 ground state of bis-ligand complexes of F430. The value of D is sensitive to the nature of the Ni(II) axial ligands: bis-aquo F430, D = +9 +/- 1 cm-1; bis-imidazole F430, D = -8 +/- 2 cm-1. Measurement of D = +10 +/- 1 cm-1 for F430 in the methylreductase holoenzyme argues strongly against histidine imidazole coordination to Ni(II) in the enzyme. The possible existence of alcoholic or phenolic oxygen-containing ligands (serine, threonine, tyrosine, water) to Ni(II) in the enzyme-bound cofactor is discussed.     

Magnetization of manganese superoxide dismutase from Thermus thermophilus.

The ground state magnetic properties of manganese superoxide dismutase from Thermus thermophilus in its native and reduced forms have been determined using saturation magnetization data. Parallel EPR measurements were used to verify that commonly encountered paramagnetic impurities were at low concentration relative to the metalloprotein. The native enzyme contains high spin Mn(III) (S = 2) with D = +2.44(5) cm-1 and E/D = 0. The reduced enzyme contains high spin Mn(II) (S = 5/2) with D = +0.50(5) cm-1 and E/D = 0.027. These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds. In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(III) present. The result, epsilon 480 = 950(80) M-1 cm-1 (delta epsilon 480 = 740(60) M-1 cm-1) agrees with the previously reported value of epsilon 480 = 910 M-1 cm-1 found by total manganese determination (Sato, S. and Nakazawa, K. (1978) J. Biochem. 83, 1165-1171). The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed.     

Structure of apoproteins in insect lipophorin

The two polypeptide chains of cockroach and locust lipophorins were separated and their amino acid compositions were determined. Circular dichroic spectra of the lipophorins and apolipophorin from 190 to 250 nm showed a single trough at 218 nm and a peak at 194 nm. Infrared spectra of the lipophorins in D2O showed a strong peak at 1625 cm-1 and a weak shoulder at 1693 cm-1 corresponding to v (pi, 0) and nu (0, pi) of antiparallel pleated sheet. The resonance frequency splitting delta nu = nu (0, pi) -nu (pi, 0) was 68 cm-1, which was larger than that of ordinary globular proteins containing antiparallel pleated sheet. From circular dichroic and infrared spectra it was concluded that lipophorins contained polypeptides rich in antiparallel pleated sheet with longer unbroken extensions than the case for ordinary globular proteins. Partial proteolytic digestion study of lipophorins with trypsin, chymotrypsin, and subtilisin showed that the larger apolipophorin (AL1) was exposed to the surface of the particle and the smaller apolipophorin (AL2) lay protected from the attack of the enzymes. Crosslinked products between AL1 and AL2 were readily obtained when dimethylsuberimidate or dimethyladipimidate was added to the lipophorin solution, without giving lipophorin dimers, suggesting that the two chains were located within 11 A from each other. Such structural features of insect lipoprotein were compared with other insect lipophorins and the human serum low-density lipoprotein (LDL). Similarities between lipophorins and LDL were found in the molecular weight, amino acid compositions, and the secondary structure of major apoproteins.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

Hydroxyl-ion-induced subunit dissociation of east cytoplasmic pyruvate decarboxylase. A circular dichroism study

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250--320-nm range a multiple two-signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are delta G = 87.7 kJ mol-1, delta H = + 56.0 kJ mol-1, delta S = - 108 J mol-1 K-1, very characteristic for the deprotonation of an amino-acid side chain. The second class of the protons has the following thermodynamic data: delta G = 88.3 kJ mol-1, delta H = - 64.3 kJ mol-1, delta S = - 520 J mol-1 K-1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. Th enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate-determining for the overall process. The data are kp = 1.4 . 10(-3), delta H not equal to = + 128.3 kJ mol-1, delta S not equal = + 136 J mol-1 K-1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor beta = kc/(1 + Kc) (0 < beta less than or equal to 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo-enzyme are derived from the data.     

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)     

Assay and purification of the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri

A continuous spectrophotometric assay was developed for the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Thereby the product (R)-3-methylitaconate is converted by the delta-isomerase from the same organism to 2,3-dimethylmaleate which absorbs at 240 nm, much higher than both parent compounds (delta epsilon = 3.7 mM-1.cm-1). In addition a discontinuous assay using the facile formation of 2,3-dimethylmaleic anhydride in aqueous solution at pH 0-1 (delta epsilon = 4.0 mM-1.cm-1 at 256 nm) was established. The mutase and the isomerase were purified together by chromatography on quaternary-amine-Sepharose (Q-Sepharose) and on cyanocobalamin-agarose. The enzymes were separated and obtained in homogenous forms by preparative PAGE in non-denaturing buffer. Both enzymes appear to be homotetramers with subunits of 70 kDa (mutase) and 50 kDa (isomerase). The equilibrium constants for both reactions were determined at I = 0.1 M and 25 degrees C: K1, app = [(R)-3-methylitaconate].[2-methyleneglutarate]-1 = 0.26 +/- 0.04, K2,app = [2,3-dimethylmaleate].[(R)-3-methylitaconate]-1 = 7.40 +/- 0.21.     

Parallel double helical DNA. 1. Evidence for the existence of a spiral A-T-paired base

The dependence of UV and CD spectra of oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-(pApTpApTpApTpApTp ApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl on temperature and concentration was studied. It was shown that the eicosamer in concentrations below 5.10(-4) M forms a parallel stranded hairpin. From the thermal denaturation profile the thermodynamic parameters of parallel hairpin formation were determined. The values of delta H0, delta S0 and Tm were -90 +/- 8 kJ/mol, -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel helix differ from those of B-form DNA by reduction of extreme magnitude at approximately 265 nm and appearance of a negative effect at approximately 285 nm.     

A resonance Raman study on the structures of complexes of flavoprotein D-amino acid oxidase

Resonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is delta 1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-alpha-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2-symmetric stretching mode of the product, such as delta 1-piperideine-2-carboxylate or delta 1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).     

The flavin dimers I. The application of absorption in anti-stokes excitation region to investigate the flavin dimer formation

The dimer formation process of the flavin in aqueous solution has been studied. The difference absorption spectra with the change of concentration in Stokes and anti-Stokes excitation region of the flavomononucleotide and riboflavin were measured. The highest temperature in which the dimers still appear is discussed. It is suggested that this temperature T_d can be treated as one of the empirical parameters which describe the dimer formation process of the dyes in solutions. The aqueous solution of flavins with the concentration c?5·10^?5 M at room temperature can be treated as a flavin monomers solution. For higher concentrations the flavin monomers and dimers exist in a solution at room temperature.     

The Type 3 copper site is intact but labile in Type 2-depleted laccase

We report results of experiments designed to characterize the Type 1 and Type 3 copper sites in Rhus laccase depleted of Type 2 copper (T2D). Use of the Lowry method for determining protein concentration yielded the value 5620 +/- 570 M-1 cm-1 for the extinction of the 615-nm absorption band of this protein. Anaerobic reductive titrations with Ru(NH)3)6(2)+ and Cr(II)aq ions established the presence of three electron-accepting centers, which are reduced in a complex manner. Treatment of T2D laccase with a 70-fold excess of H2O2 induced a new shoulder at 330 nm (delta epsilon = 660 M-1 cm-1), as well as intensity perturbations at 280 and 615 nm. Comparison of difference spectra show that this 330-nm band derives from a Type 3 copper-bound peroxide and not from a reoxidized Type 3 site. Dioxygen reoxidation of ascorbate-reduced T2D laccase produced new difference bands at 330 nm (delta epsilon = 770 M-1 cm-1) and 270 nm (delta epsilon = 13,000 M-1 cm-1), the former assigned to a bound peroxide which is a dioxygen reduction intermediate. In the corresponding epr spectrum of this material new Cu(II) g parallel features (A parallel approximately 130 G) indicative of an isolated copper ion and a triplet signal near 3,400 G were observed, originating from the Type 3 sites of separate T2D laccase molecules. Reoxidation by ferricyanide or by dioxygen as mediated by iron hexacyanide did not produce these changes. Thus the magnetism of the reoxidized Type 3 site in T2D laccase can be perturbed as a consequence of aerobic turnover. The suggestion is advanced that there are presently three forms of T2D laccase, possibly metastable conformational isotypes, accounting for the apparently contradictory reports on the properties of this protein.     

Pathways of electron transfer photosensitized by thiacyanine dimers

Pathways of electron transfer reaction between p-nitroacetophenone (p-NAP) and ascorbic acid (AA) photosensitized by dimers of 3,3'-disulfopropyl-5,5'-dichlorothiacyanine triethylammonium (TC) and 3,3'-disulfopropyl-5,5'-dichloro-9,11-[ββ-dimethyltrimethylene]thiadicarbocyanine triethylammonium (TDC) are considered. In aqueous solution the dyes are present as an equilibrated mixture of monomers (M(-)) and dimers (M(2)(2-)). In contrast to monomers, the dimers of TC are characterized by a noticeable yield of intersystem crossing, whereas for TDC the triplet-triplet absorption of both monomers and dimers is easily observed upon ns-laser pulse. In the presence of p-NAP and AA the triplet state of the dimers of both dyes is mostly quenched by p-NAP yielding the radical pair [M(2)(-)˙p-NAP(-)˙] with subsequent dissociation of M(2)(-)˙ into M(-) and M˙ followed by one-electron reduction of M˙ by AA. These steps constitute a pathway of photosensitization by the dimers. For TDC an additional pathway of photosensitization was found to occur. The primary step consists of electron transfer in the excited singlet state of the dimer resulting in the formation of the radicals M˙ and M(2-)˙. The next steps involve one-electron reduction of M˙ by AA and one-electron oxidation of M(2-)˙ by p-NAP which results in the formation of M(-) followed by dimerization.     

Liposome formation of egg lecithin and its interaction with iodine

The sonicated dispersion of egg lecithin (phosphatidylcholine) in water forms 1:1 molecular complex with iodine, when its concentration is above 1.6 X 10(-5) M. The thermodynamic and spectrophotometric properties of this complex have been determined. The thermodynamic values are: K (25 degrees C) = 1.6 X 10(3) 1 X mol-1, delta G degrees = -18.4 KJ X mol-1, delta H degrees = -27.4 KJ X mol-1 and delta S degrees = -30.0 J X mol-1 X deg-1. The complex shows two absorption bands: one at 293 nm, which is the charge transfer band and the other at 370 nm, which is the blue shifted visible iodine band at 460 nm in water.     

Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo).

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with 'high affinity' (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with 'low affinity' (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.