Purification and properties of hydrogenase from phototrophic bacterium Thiocapsa roseopersicina]

The isolation method and some peoperties of purple sulphur bacteria (Thiocapsa roseopersicina strain BBS) hydrogenase are described Hydrogenase molecular weight is found to be 66000; it contains 3.7 moles of S2- and 3.9 moles of Fe2+ per one mole of the enzyme;pI=4.2. The enzyme absorption spectrum has the maximum at 400-412 nm which is characteristic of proteins containing non-haem iron. Hydrogenase is suggested to consist pf 4 subunits of two types: with molar weight 27000 and 6000. Unlike other hydrogenases, this enzyme is rather resistant to O2 and is more thermostable: the inactivation of the enzyme was observed at the temperature above 80 degrees C; Hydrogenase preparation catalyses D2-H2O exchange reaction, H2 evolution from the reduced methyl viologene (MV) and H2 absorption in the presense of MV or benzylviologene but not in the presense of NAD(P), FAD, FMN, azocarmine, methylene blue and ferricyanide.     

The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China

The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity, deoxyribonucleic acid (DNA) under isothermal condition in simple incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of 18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic DNA of two Babesia species, respectively, and did not cross-react with the genomic DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of DNA present in 50 μl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore, DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial infection in sheep and goats.     

Relaxation kinetics of cytochrome P450 reductase: internal electron transfer is limited by conformational change and regulated by coenzyme binding

The kinetics of internal electron transfer in human cytochrome P450 reductase have been studied using temperature-jump relaxation spectroscopy. Temperature perturbation of CPR reduced at the two-electron level with NADPH yields biphasic absorption transients at 450 and 600 nm. The observed rate, 1/tau, for the fast phase is 2200 +/- 300 s(-1). The absence of this phase in fluorescence transients and in absorption transients collected with dithionite-reduced enzyme indicates this phase does not report on electron/hydride transfer and is consistent with its origin in local conformational change in the vicinity of the FAD isoalloxazine ring. The slow phase (1/tau = 55 +/- 2 s(-1)) observed in the absorption transients obtained with CPR reduced at the two-electron level with NADPH reports on internal electron transfer: FAD(sq)-FMN(sq) --> FAD(ox)-FMN(hq). The observed rate of this transient is slower (1/tau = 11 +/- 0.5 s(-1)) in CPR reduced to the two-electron level by dithionite rather than NADPH, demonstrating that coenzyme binding has an important influence on the observed rate of internal electron transfer. Temperature perturbation experiments with CPR reduced with 10-fold molar excess of NADPH produce monophasic absorption transients (1/tau = 20 +/- 0.2 s(-1)) reporting on internal electron transfer: FAD(sq)-FMN(hq) --> FAD(hq)-FMN(sq). The observed rate constants for electron transfer are substantially less than those expected from analysis of CPR by electron-transfer theory (approximately 10(10) s(-1)). Potential gating mechanisms have been investigated using the temperature-jump method. Observed rates for electron transfer were unaffected in experiments performed in deuterated solvent, indicating that deprotonation does not gate the reaction. Introduction of glycerol into the sample significantly decreased the observed rate for internal electron transfer, suggesting conformational gating of the reaction. Replacement of Trp-676 with His-676 reduces approximately 2-fold the observed rate of internal electron transfer in two-electron-reduced enzyme, whereas the observed rate for FAD(sq)-FMN(hq) --> FAD(hq)-FMN(sq) transfer is increased approximately 13-fold in the W676H mutant reduced with a 10-fold molar excess of NADPH. The studies reveal altered redox properties of the FAD in W676H CPR. The data are discussed in the context of previous stopped-flow studies of human CPR and the X-ray crystallographic structure of rat CPR.     

Regulation of PKM2 and Nrf2-ARE Pathway during Benzoquinone Induced Oxidative Stress in Yolk Sac Hematopoietic Stem Cells

Benzene is an occupational toxicant and an environmental pollutant that is able to induce the production of reactive oxygen species (ROS), causing oxidative stress and damages of the macromolecules in target cells, such as the hematopoietic stem cells. We had previously found that embryonic yolk sac hematopoietic stem cells (YS-HSCs) are more sensitive to benzene toxicity than the adult bone marrow hematopoietic stem cells, and that nuclear factor-erythroid-2-related factor 2 (Nrf2) is the major regulator of cytoprotective responses to oxidative stress. In the present report, we investigated the effect of PKM2 and Nrf2-ARE pathway on the cellular antioxidant response to oxidative stress induced by benzene metabolite benzoquinone (BQ) in YS-HSC isolated from embryonic yolk sac and enriched by magnetic-activated cell sorting (MACS). Treatment of the YS-HSC with various concentrations of BQ for 6 hours induces ROS generation in a dose-dependent manner. Additional tests showed that BQ is also capable of inducing expression of NADPH oxidase1 (NOX1), and several other antioxidant enzymes or drug-metabolizing enzymes, including heme oxygenase 1 (HMOX1), superoxide dismutase (SOD), catalase and NAD(P)H dehydrogenase quinone 1 (NQO1). Concomitantly, only the expression of PKM2 protein was decreased by the treatment of BQ but not the PKM2 mRNA, which suggested that BQ may induce PKM2 degradation. Pretreatment of the cells with antioxidant N-acetylcysteine (NAC) decreased ROS generation and prevented BQ-induced PKM2 degradation, suggesting involvement of ROS in the PKM2 protein degradation in cellular response to BQ. These findings suggest that BQ is a potent inducer of ROS generation and the subsequent antioxidant responses of the YS-HSC. The accumulated ROS may attenuate the expression of PKM2, a key regulator of the pyruvate metabolism and glycolysis.     

Antagonism of endothelin-1 inhibits hypoxia-induced apoptosis in cardiomyocytes

Apoptosis is well documented to be a common feature of many pathological processes of the heart. Exogenous endothelin-1 (ET-1) has been shown to be proapoptotic or antiapoptotic, depending on ET-1 concentration, cell type, and the ratio of ETA/ETB receptor subtypes. The role of endogenous ET-1 in cardiomyocyte apoptosis, however, is not clarified. This study observed the effects of the ETA-receptor antagonists BQ610 and BQ123 and the ETB-receptor antagonist BQ788 on hypoxia-induced apoptosis in primary cultured neonatal rat cardiomyocytes. Hypoxic apoptosis was induced by incubating cardiomyocytes in serum-free medium under 3% O2 and 5% CO2 for 24 h and evaluated by TUNEL analysis and flow cytometry. TUNEL analysis showed that the apoptotic cardiomyocytes constituted 24.2% +/- 2.2% of the total cells under hypoxic conditions. Treatment with BQ610 (5 micromol/L) significantly reduced the apoptosis rate to 13.2% +/- 3.7% (data from 4 independent experiments, p < 0.01 vs. hypoxia). Flow cytometry showed that the percentage of apoptotic cells positively stained with annexin V and propidium iodide was 42.76% +/- 4.44% (n = 12) in cultures subjected to hypoxia. BQ123 at 0.04, 0.2, and 1.0 micromol/L dose-dependently reduced the apoptosis rate to 34.00% +/- 10.35% (n = 6, p < 0.05), 30.38% +/- 8.28% (n = 6, p < 0.01), and 22.89% +/- 4.19% (n = 6, p < 0.01), respectively. In contrast, BQ788 did not affect hypoxic apoptosis. These findings suggested that endogenous ET-1 contributed to hypoxia-induced apoptosis in cultured cardiomyocytes, which was mediated by ETA receptors, but not by ETB receptors.     

Niacin skin-flush response and electrodermal activity in patients with schizophrenia and healthy controls

Patients with schizophrenia have in different studies shown reduced niacin sensitivity and lower electrodermal activity (EDA) after auditory stimulation. Peripheral mediation of prostaglandins may have a physiological role in both responses. This motivates study of both niacin response and electrodermal responding in the same patients with schizophrenia. Thirty patients with schizophrenia and 17 controls were investigated with EDA and thereafter given 200mg niacin orally with continuous assessment of skin temperature. The patients showed a delayed temperature increase after niacin ingestion (P=0.002) and a higher frequency of electrodermal non-responding (P<0.05). Response/non-response for niacin correlated with EDA response/non-response in the patient group (P=0.009). The niacin test revealed a slower vasodilation reaction in the patients. The association between response patterns for the niacin test and EDA suggests that a common aberration in skin physiology may be of importance for both reactions in schizophrenia.     

An enzymatic colorimetric method for measuring naringin using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) in the presence of peroxidase

A sensitive colorimetric method for naringin estimation using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as peroxidase substrate is described. The method is based on the coupling reaction of an ABTS radical cation with an oxidation product of naringin formed by peroxidase. This coupling reaction leads to the formation of a purple-colored compound with a maximum absorption at 560 nm. A molar absorption coefficient at this wavelength was calculated to be 13,286 +/- 200 M-1 cm-1. The lowest amount of naringin that can be detected is 1 nmol. The application of this method to the quantification of naringin in grapefruit tissues is presented.     

The binding of a thyroid hormone metabolite, 3-monoiodo-L-thyronine, to bovine serum albumin as measured by circular dichroism

The interaction between a thyroid hormone metabolite, 3-monoiodo-L-thyronine (3-T1) and bovine serum albumin (BSA) was investigated by using the CD method. An enhanced CD band was observed at the absorption wavelength region of 3-T1 around 293 nm suggesting the binding of 3-T1 to the BSA molecule. The ellipticity at 293 nm was measured at various molar ratios of 3-T1 to BSA, and the apparent binding constant and the maximum number of binding sites could be estimated as Kapp = 8.85 +/- 1.07 X 10(4) M-1 and n = 23.8 +/- 0.9 respectively. The CD of a mixture of BSA, 3-T1 and thyroxine (T4) was also studied at various pH's. The pH profile of the two characteristic CD bands at 293 nm and 320 nm, attributed to bound 3-T1 and T4, suggested that the optimum binding condition of 3-T1 was attained at alkaline pH of around 9, while that of T4 was attained over a wide pH range between 5-10. A significant role of the ionized 4'-hydroxyl group of 3-T1 in the binding reaction with BSA is also suggested.     

Molar absorption coefficients for the reduced Ellman reagent: reassessment

The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.     

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients.

Two independent sets of measurements using a variety of methods show that a sample with A_{800 nm} = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1.

Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77.

These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: _{802 nm} = 288 (± 14) mM⁻¹ · cm⁻¹ and _{865 nm} = 128 (± 6) mM⁻¹ · cm⁻¹. For reversible bleaching of the 865-nm band, Δ^{red - ox}_865nm = 112 (± 6) mM⁻¹ · cm⁻¹ (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

Inhibition of jack bean urease by 1,4-benzoquinone and 2,5-dimethyl-1,4-benzoquinone. Evaluation of the inhibition mechanism

1,4-benzoquinone (BQ) and 2,5-dimethyl-1,4-benzoquinone (DMBQ) were studied as inhibitors of jack bean urease in 50 mM phosphate buffer, pH 7.0. The mechanisms of inhibition were evaluated by progress curves studies and steady-state approach to data achieved by preincubation of the enzyme with the inhibitor. The obtained reaction progress curves were time-dependent and characteristic of slow-binding inhibition. The effects of different concentrations of BQ and DMBQ on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. The rapid formation of an initial BQ-urease complex with an inhibition constant of Ki = 0.031 mM was followed by a slow isomerization into the final BQ-urease complex with the overall inhibition constant of Ki* = 4.5 x 10(-5) mM. The respective inhibition constants for DMBQ were Ki = 0.42 mM, Ki* = 1.2 x 10(-3) mM. The rate constants of the inhibitor-urease isomerization indicated that forward processes were rapid in contrast to slow reverse reactions. The overall inhibition constants obtained by the steady-state analysis were found to be 5.1 x 10(-5) mM for BQ and 0.98 x 10(-3) mM for DMBQ. BQ was found to be a much stronger inhibitor of urease than DMBQ. A test, based on reaction with L-cysteine, confirmed the essential role of the sulfhydryl group in the inhibition of urease by BQ and DMBQ.     

Photoreactivating enzyme from Escherichia coli: appearance of new absorption on binding to ultraviolet irradiated DNA.

The photoreactivating enzyme, PRE, monomerizes pyrimidine dimers in DNA in a light requiring reaction (lambda greater than 300 nm). However, the purified PRE from E. coli has no well-defined absorption band for lambda greater than 300 nm. Using absorption difference spectroscopy, we show that when PRE is mixed with ultraviolet-irradiated DNA, new absorption appears in the spectral region required for catalysis. There is a concomitant decrease in the absorption of the mixture for wavelength less than 300 nm. The hyperchromicity for lambda greater than 300 nm is true absorption, not an artifact due to light scattering. Both the hyperchromicity (lambda greater than 300 nm) and hypochromicity (lambda less than 300 nm) can be reversed by irradiation of 365 nm with identical first-order kinetics. We estimate the molar extinction coefficient of the new absorption to be 6900 +/- 1400 at 350 nm. We conclude that the PRE from E. coli does not possess a distinct "chromophore" which by itself is entirely responsible for the absorption of photoreactivating light. Instead, new absorption results when PRE binds its substrate, dimer-containing DNA.     

New spectrophotometric method for continuous recording of the spleen exonuclease activity

Some of the synthetic chromophoric substrates of various enzymes cannot be used for direct spectrophotometric recording of the reactions, when a difference between the pH optimum of the enzyme reaction and the pH of maximum absorption of the released chromophore exists. In the present paper we describe a new method for following the time course of the spleen exonuclease-catalyzed reaction with thymidine 3'-monophospho-p-nitrophenyl ester as a substrate, based on the difference obtained in the absorbency of the substrate and its products in the far UV (at 330 nm). This difference, not published before, permits direct spectrophotometric recording of the amount of the hydrolyzed chromophoric substrate in acidic pH, whereas the maximum absorption of the product as accepted in the literature, is in alkaline pH. The molar absorption coefficient of the measurement at pH 5.7 is determined to be epsilon = 522 M-1.mm-1.     

The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite

The first-derivative EPR spectrum of the active form of Escherichia coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site. The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formate-lyase which destroys the enzyme radical. Protein-linked 1-hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445-450]. EPR spectroscopy of this system has now identified the corresponding alpha-phosphoryl radical as a reaction intermediate [g = 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0 degrees C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.     

Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

The anti-malarial drug quinine and its quaternary derivative N-benzylquininium (BQ(+)) have been shown to inhibit gap junction (GJ) channels with specificity for Cx50 over its closely related homologue Cx46. Here, we examined the mechanism of BQ(+) action using undocked Cx46 and Cx50 hemichannels, which are more amenable to analyses at the single-channel level. We found that BQ(+) (300 μM-1 mM) robustly inhibited Cx50, but not Cx46, hemichannel currents, indicating that the Cx selectivity of BQ(+) is preserved in both hemichannel and GJ channel configurations. BQ(+) reduced Cx50 hemichannel open probability (P(o)) without appreciably altering unitary conductance of the fully open state and was effective when added from either extracellular or cytoplasmic sides. The reductions in P(o) were dependent on BQ(+) concentration with a Hill coefficient of 1.8, suggesting binding of at least two BQ(+) molecules. Inhibition by BQ(+) was voltage dependent, promoted by hyperpolarization from the extracellular side and conversely by depolarization from the cytoplasmic side. These results are consistent with binding of BQ(+) in the pore. Substitution of the N-terminal (NT) domain of Cx46 into Cx50 significantly impaired inhibition by BQ(+). The NT domain contributes to the formation of the wide cytoplasmic vestibule of the pore and, thus, may contribute to the binding of BQ(+). Single-channel analyses showed that BQ(+) induced transitions that did not resemble pore block, but rather transitions indistinguishable from the intrinsic gating events ascribed to loop gating, one of two mechanisms that gate Cx channels. Moreover, BQ(+) decreased mean open time and increased mean closed time, indicating that inhibition consists of an increase in hemichannel closing rate as well as a stabilization of the closed state. Collectively, these data suggest a mechanism of action for BQ(+) that involves modulation loop gating rather than channel block as a result of binding in the NT domain.     

Adjusted 200-day scrotal size as a predictor of 365-day scrotal circumference

Data collected from 264 bulls of 13 beef breeds at the Michigan Bull Test Station was evaluated to determine if scrotal circumference (SC) adjusted to 200 d of age could be used to predict scrotal circumference at 1 yr of age. Scrotal circumference of each bull was recorded on arrival at the test station and at the time of breeding soundness examination (BSE) and was adjusted to 200 and 365 d of age, respectively. Bulls with adjusted SC>34.0 cm by 365 d of age averaged a larger SC at 200 d (P < 0.0001) and faster scrotal growth (P < 0.0001) than bulls with a 365 d adjusted SC /= 23.0 cm had a 95% probability of achieving SC > 34.0 cm by one year of age. Calves measuring < 23 cm at 200 d had a 54% probability of achieving > 34.0 cm scrotal size by one year. This information can contribute to the selection of breeding bulls that will achieve desirable scrotal size by one year of age.     

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.     

Structure and thermal behavior of a cellulose I-ethylenediamine complex

We prepared highly crystalline samples of a cellulose I-ethylenediamine (EDA) complex by immersing oriented films of algal (Cladophora) cellulose microcrystals in EDA at room temperature for a few days. The unit-cell parameters were determined to be a = 0.455, b = 1.133, and c = 1.037 nm (fiber repeat) and gamma = 94.02 degrees. The space group was P2(1). On the basis of unit cell, density, and thermogravimetry analyses, the asymmetric unit is composed of one anhydrous glucose residue and one EDA molecule. The chemical and thermal stabilities of the cellulose I-EDA complex were also investigated by the use of X-ray diffraction. When the cellulose I-EDA complex was immersed in methanol or water at room temperature, cellulose III I or I beta was obtained, respectively. However, immersion in a nonpolar solvent such as toluene did not affect the crystal structure of the complex. The cellulose I-EDA complex was stable up to a temperature of approximately 130 degrees C, whereas the boiling point of EDA is 117 degrees C. This thermal stability of the complex is probably caused by intermolecular hydrogen bonds between EDA molecules and cellulose. When heated above 150 degrees C, the cellulose I-EDA complex decomposed into cellulose I beta.