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In this paper equilibrium models for the calculation of the excess Gibbs free energy of binary liquid mixtures are developed, the component A of which undergoes chain-forming self-association whilst the component B acts as an 'inert' solvent. It is shown that the extension of the well-known chain-association model of Mecke and Kempter, in which the probability of chain prolongation is assumed to be independent of chain length, is unable to establish satisfactory results because it does not exhibit sufficient unsymmetry. Reduction of the probability of chain growth with in-creasing chain length leads to an improved model with the geometric series replaced by the exponential series. This model, in which only two parameters are used, i. e. the equilibrium constants K for mutual solvation of A and B, and ρ for self-association of A, allows fitting of isothermal experimental GE /R T literature data on cycloalkanol-cycloalkane, alkanol-alkane, and NMF -CCl4 systems within the limits of experimental error. Compared with the two-parameter Wilson equation which gives equally small standard deviations, our equilibrium model has the advantage of allowing passage from GE to HE data and of being applicable to liquid-liquid equilibria.
Nuclear magnetic resonance measurements were carried out on neutron activated 20F(T1/2=11s) nuclei in a single crystal of KZnF3. The quadrupolar splitted NMR spectrum, detected via the 20F β-radiation asymmetry, could be observed using a radio frequency modulation technique. The quadrupole coupling constant was determined to e2 q Q/h= + (12.0 ± 1.5) MHz at room temperature. The sign of e2 q Q was obtained from a simultaneous γ-ray anisotropy measurement on the succeeding 20Ne transition. Utilising a calculated field gradient of the fluorine atom, an fQ = 4.6% is determined. This value is compared with literature data of similar compounds.
Diadamantyldioxetane, trim ethyldioxetane and tetram ethyldioxetane were photolyzed b y light of A > 260 nm . The spectral distribution o f the quanta emitted during photoinduced decom position of dioxatenes was found to be different from fluorescence and phosphorescence o f ketones. Flash photolysis experim ents showed the absorption of an short-lived interm ediate. It was concluded, therefore, that photolysis o fdioxetanes is not a concerted process but involves at least one precursor o f the final product ketone.
ncubation of class II chloroplasts of spinach with copper in the light at pH = 8 in concentrations that inhibit oxygen evolution results in the formation of a copper (II) protein complex with the photosynthetic membrane. The EPR spectra indicate that the four nearest ligands to Cu(II) consist of three oxygen atoms and one nitrogen atom. The copper (II) protein appears to be pre dominantly associated with photosystem II. The formation of this protein as measured by the EPR signal amplitude of its room temperature spectrum correlates with the inhibition of oxygen evolution and of electron transport within photosystem I. This result indicates that the inhibition of photosynthetic electron transport by copper may be due to the formation of a copper (II) chelate with a membrane protein.
The thermal decomposition of 1,2-diadamantyldioxetane was studied by kinetic and spectroscopic methods. Spectra of the chemiluminescence emitted during the thermally induced decomposition of 1,2-diadamantyldioxetane, tetramethyldioxetane and trimethyldioxetane were obtained and the influence of quenchers and radical-scavengers, and the presence of "heavy atoms" in the surrounding of the emitting species was investigated. The kinetics of the decay mechanism was followed by measuring the time dependence of the chemiluminescence. The influence of radical-scavengers, quenchers and "external heavy atoms" on the kinetics was assessed. Experimental results were discussed in terms of a biradical decay mechanism.
Intoxication of class II chloroplasts of spinach with Cu(II) leads to inhibition of millisecond luminescence. The degree of inhibition depends on Cu (II) -concentration. The investgation of the pH dpendence of the inhibition curve of luminescence revealed that (1) there is an inhibition site of copper on the donor side of photosystem II, (2) copper (II) does not act as an uncoupler of photophosphorylation, (3) a protonation equilibrium is involved in the inhibition mechanism, and (4) copper (II) binds to a dissociated residue of a membrane protein.
Some physical and chemical properties of the cancerostat cyclophosphamide (generic name: ENDOXAN) and its basic constituents H3PO4 and nor-N-mustard have been calculated with the help of a modified CNDO/S-method. The spectroscopic data of the H3PO4 , which is the starting-point for a corresponding calculation of cyclophosphamide, has been studied by taking account of the 3 d electron of the phosphorus. Nor-N-mustard is a very reactive compound, characterized by the ability to split off chloride ions and to act as an alkylating agent. The binding of the nor-N-mustard to the cyclic phosphate ester (cyclophosphamide) modifies the chemical reactivity of the mustard group in an essential way, and the 3d electron of the phosphorus plays an important role with respect to the excitability of the C -Cl bonds. Cyclophosphamide must be metabolized in a suitable way to develop the same alkylating activity as the nor-N-mustard. The computation of the excited states of cyclophosphamide revealed a similar term scheme as it was found by Clar in the case of the carcinogenic polycyclic hydrocarbons.
3,17 β-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol of Streptomyces hydrogenans. After ammonium sulfate precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to molecular weight of 70 200 ± 2 500. 3 β-. 17 β- as well as 20 β-hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accent NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 μᴍ for 5 α-dihydrotestosterone, 20 μᴍ for testosterone ana 68 μᴍ for epiandrosterone in the NAD+-driven reaction, 1.8 x 10-4 m for NADH+ and 1.9 x 10-4 ᴍ for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki 1.15 x 10-3 ᴍ).
After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing column (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 - 5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the figure inceases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially.
The effect of NH4Cl on the kinetics of the back reaction of photosystem II as derived from luminescence measurements was investigated in dark adapted Chlorella in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at different temperatures. The kinetics of the back reaction which, under these conditions, leads to the reduction of the S2 state by the primary electron acceptor Q- of photosystem II was observed to be considerably slowed down in the presence of NH4Cl.
Analysis of the kinetic results in the light of the theory of the back reaction developed by Mar and Roy (J. Theor. Biol. 48, 257-281 (1974)) revealed two opposite effects of NH4Cl to be present simultaneously:
1) The enthalpy of activation of the back reaction was lowered (catalyzing effect of NH4Cl)
2) The frequenca factor which indicates the number of collisions of the reacting molecules in the membrane per second is largely decreased (inhibitory effect of NH4Cl).
This reduction of the mobility of the recombining species of the back reaction is the predominant effect of NH4Cl. It is suggested that this effect is due to a change of the conformational state of the membrane induced by dissolution of relative large amounts of NH? within the lipid phase of the thylakoid membrane. This hypothesis is supported by the observation that the value of the exciton yield of the back reaction changes upon addition of NH4Cl.
Photosystem II
It is shown that the kinetics of the back reaction of photosystem II in the seconds time range as derived from the luminescence decay curve in the presence of DCMU is controlled by the internal pH of the thylakoids. Modifications of the conformational state of the photosynthetic membrane while leaving the internal pH unchanged, however, reaction.
Methods are described for an enzymatic preparation of 14C-labeled terpenoids. With a cell-free system of a white mutant of Phycomyces blakesleeanus (Mucoraceae) [14C]squalene and [14C- cis]phytoene can be synthesized from [2-14C]mevalonate. The application of norflurazon, a phenyl- pyridazinone herbicide, helps to increase the yield of squalene. Furthermore, the liquid endosperm of Echinocystis lobata (Cucurbitaceae) was used for the formation of either [14C(-)]kaurene from [14C]mevalonic acid or [14C-/ra/w]geranylgeranyl pyrophosphate in the presence of Amo 1618.
The hydrocarbons formed were purified by alumina-column chromatography and preparative thin-layer chromatography (TLC). Geranylgeranyl pyrophosphate was separated by DE-column chromatography followed by TLC.
Potential energy and dipole moment functions have been calculated for the ground states of the NeH+ (1.0 ≦ R ≦ 15 a. u.) and the KrH+ (1.6 ≦ R ≦ 20 a. u.) ion from highly correlated SCEP/VAR and SCEP/CEPA electronic wave functions. The following spectroscopic constants have been derived: Ne20H+ re = 0.996 ± 0.003 Å, ωe = 2896 ± 20cm-1 , D0(Ne + H+) = 2.10 ± 0.05 eV; Kr84H+ re = 1.419 ± 0.003 Å, ωe = 2561 ±20 cm-1 , D0(Kr + H+) = 4.65 ±0.05 eV. The Einstein transition probability coefficients of spontaneous emission have been calculated for all transitions v ≦ 5 of Ne20H+, Ne20D+, Kr84H+ and Kr84D+, respectively.
For a certain class of ocean models describing the exchange of inorganic carbon between the atmosphere and the surface layer of the ocean as well as between the surface layer and the deep sea the dynamical airborne fraction is evaluated analytically under the assumption that the growth rate of the atmospheric source term (fossil fuel plus net biogenic carbon input into the atmosphere) is slowly variable with time. Each of these models exhibits a certain uptake capacity of the deep ocean which is quantified. Considerations are made as to whether the terrestrial biota are to be regarded as a source or a sink for additional atmospheric CO2 depending on the modelling of the deep ocean. It is shown that a global one-dimensional box-diffusion ocean model with a depth dependent eddy diffusivity K(z) - K(0) exp[-z/z*], with an adjustable parameter set {K(0), z*}, provides a fairly well fit to the prebomb 14C ocean distribution and to an appreciable net biogenic carbon transfer into the atmosphere. The range of future atmospheric CO2 partial pressures is estimated for a given fossil input.
Membrane-Phloretin Interaction, Infrared Raman, ESR Spectroscopy The transport inhibitor phloretin was bound to human red cell membrane and the concomitant structural changes were observed by spectroscopic methods. By the spin labeling method a decrease in fluidity of the membrane was found at 1 and 10 |iM concentrations of the reagent. This result was obtained with the 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, and the 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl lipid spin labels. Infrared spectroscopy of modified membranes revealed an intensity increase of the POO~ band at about 1250 cm-1. Moreover, a shift of the peak at 1050 cm -1 to 1100 cm-1 was observed in the presence of phloretin. Raman spectroscopy of the membranes did not contradict the results found with infrared and ESR spectroscopy: In the phloretin modified membrane we observed a lack of the band at 1085 cm-1, which leads to suggest that the POO" and/or C-C regions are less fluid. Changes of the extracted red cell membrane lipids were less characteristic, and the results differed from those found in red cell membrane.
The influence of temperatur and pressure on the fluorescence quantum yield of N-methylacridone (9,10-dihydro-9-oxo-10-methyl-acridine) in toluene in the range of 283-313 K and 1 bar to 2.5 kbar, respectively, has been investigated. Treatment of the data in terms of the Eyring transition-state theory leads to a consistent interpretation of the observed effect. The unusually large increase of the quantum yield with increasing pressure is attributed to a positive volume of activation, ⊿V≠, for the thermally activated S1-T2 intersystem crossing which is known to be the only deactivation process (of the Si-state) competing with fluorescence. Comparison of the values for ⊿H≠, the activation enthalpy of this process, determined at various pressures, indicates a decrease in ⊿H≠ at elevated pressures. Since ⊿H≠ can be associated with the S1-T2 energy gap involved in intersystem crossing, this result further confirms the conclusion that the change in Franck-Condon factors alone cannot account for the decrease in the intersystem crossing rate with increasing pressure.
The crystal structure of the low temperature phase of anilinium bromide, C6H5NH3⊕Br⊖, was studied by neutron diffraction at T = 100 K. The refinement supports an ordered structure. The structures of the low and high temperature phases are compared and the mechanism of the phase transformation is discussed.
The application of laser induced fluorescence (LIF) in the uv to monitor tropospheric OH concentrations is limited for several reasons. In general the sensitivity of this method increases with the laser intensity. But at the low OH concentrations present in the atmosphere the beginning nonlinearity of the absorption (saturation effect) severely restricts the use of higher laser intensities. The high sensitivity of the LIF technique can be further compromised by the presence of an OH interference signal. This signal is generated by the monitoring laser light itself from laser photolysis of ambient ozone and the succeeding reaction of the photolysis product O (1D) with water to produce hydroxyl radicals. The results of the calculations are presented in a diagram from which the range of laser parameters can be deduced, which can be applied with confidence to monitor OH by the LIF method. The maximum number of signal counts for these working conditions is in the range of 10-3 per laser pulse.
The crystal structure of the high temperature phase of anilinium bromide, C6H5NH3⊕Br⊖ , was studied by X-ray and neutron diffraction at T = 343 K. The refinement supports disordered positions of the -NH3⊕ group. A split-atom model is proposed which includes disorder of the benzene ring. The thermal parameters, hydrogen bond distances, and other experimental data (NMR, NQR, inelastic neutron scattering) are in accordance with this model.
pH-titrations with NADH show two ionizable groups in mitochondrial and cytoplasmic malate dehydrogenase, the first with a pKa in the range 6.8 -8.3 for the mitochondrial and 6.4-7.8 for the cytoplasmic enzyme, the second with a lower limit at 10.2 resp. 11. Comparison with bis-(dihydronicotinamide)-dinucleotide and dihydronicotina-mide-ribosyl-P2-ribose-pyrophosphate instead of NADH indicates that the second alkaline ionization is caused by a residue placed near the adenine binding site of the active centre of the two isoenzymes. Binding studies with NADH and NAD+ give evidence for the participation of a group in the mitochondrial enzyme with pKa 6.8, deprotonation of which is necessary for detectable association of NAD+. In contrast the fixation of NAD+ to the cytoplasmic enzyme is independent of pH.
Stimulated emission from chemically formed excited iodine molecules has been observed. The emission originates from the vibrational state ν′ = 55 of I2(B 3 II). The excited molecules are produced by a three body recombination reaction.
The photochemical cleavage of the endoperoxides of anthradichromene and benzodixanthene into the parent hydrocarbon and oxygen is shown to be an adiabatic photoreaction originating from an upper excited singlet state. This photochemical behaviour is described by a theory for the photochemistry of endoperoxides developed by Kearns and Khan.
Influence of rotational relaxation on tropospheric OH laser induced fluorescence measurements
(1982)
Rotational relaxation of OH molecules in the 2II electronic ground state has been observed to occur in collisions with water molecules with gas kinetic probability. It causes an additional contribution to the already well known sources of interference when LIF is used to monitor tropospheric OH. As the laser generated OH is originally produced mostly in high rotational states, the fast relaxation phenomenon leads to a further population of OH in low rotational states. These states are used to monitor tropospheric OH by spectroscopic methods. The observed effect therefore increases the interference. A mathematical analysis is presented, revealing the effect of all relevant parameters.
The crystal structure of C12H11N2SiCl3 (monoclinic, P21/m, Z = 2, with a: 9.284(4), b: 7.226(2), c: 10.832(5) Å, β = 115.14(3)°) was refined to R(F) =0.035 from 1228 independent reflections. A trigonal bipyramidal, pentacoordinate silicon is observed. The chelated complex shows two different Si−N bonds, a coordinative bond (1.984(2) Å) between Si and N on the axial position and a Si−N single bond (1.737(3) A, equatorial plane), introduced by chemical reaction. The coordinative bond is 14.2% longer than the Si−N single bond. The lengthening of the coordinative bond in the present case is compared with distances in other extracoordinated silicon compounds.
Studies on the transport of anions and zwitterions of acidic amino acids in Streptomyces hydrogenans
(1983)
n Streptomyces hydrogenans, acidic amino acfds are taken up either as anions by a specific transport system or as zwitterions via a nonspecific one. Variations in the zwitterion concentration caused by changes in pH influence the uptake and exchange diffusion by the nonspecific system. Differences in pH-optima for ʟ-glutamate and ʟ-aspartate transport are due to the different pK2-values of these amino acids. The anion transport by the specific system is accompanied by a short hyperpolarization of the membrane potential followed by a secondary influx of potassium ions into the cells.
The order-disorder phase transition which occurs at Tc = 296.9 K in anilinium bromide, C6H5NH3Br, is studied by inelastic neutron time-of-flight measurements. Data are taken from hydrogenated and partially deuterated samples (C6D5NH3Br and C6H5ND3Br) below and above the transition temperature. From the inelastic scattering data the phonon frequency distribution in the range of the intermolecular vibrations is calculated. The assignment of bands is given and changes in the spectra with temperature are discussed. The analysis of the quasielastic line revealed substantial broadening due to rotational diffusion of the -NH3⊕ group. These jump diffusion processes were observed in the orthorhombic high temperature and - with markedly diminished rate - in the monoclinic phase below Tc. From the correlation times at different temperatures the activation energies are estimated for both phases. Comparison with calculated elastic incoherent structure factors suggests jumps of 60° for hindered rotation.
[4-(3-Bromoacetylpyridinio)-butyl]adenosine pyrophosphate as a structural analog of NAD+ reacts covalently with the sulfhydryl groups of thiopropyl agarose. 10-20 μmol can be bound to 1 ml gel. Stabilization of the insoluble coenzym e is attained by treatment with sodium boro hydride (NaBH4). This complex when applied to column chromatography, allow s the separation of various dehydrogenases as a result of their different complex stability coefficients. Alcohol dehydrogenase from liver, lactate dehydrogenase, and adenylate kinase, which all bind to the ADP-analog residues of the gel matrix, can thus be separated by different salt gradients. Alcohol dehydrogenase from yeast, however, does not form a complex and can easily be eluted from the column with phosphate buffer. Glyceraldehyde-3 phosphate and aldehyde dehydrogenases can be eluted by the addition of NAD+ or NADH to the buffer. The uncharged 1,4-dihydropyridin ring of the reduced coenzyme produces a more stable complex with the dehydrogenases than the oxidized form.
Near equilibrium potential energy and dipole moment functions have been calculated for the electronic ground state of the XeH+ ion from highly correlated SCEP/CEPA electronic wavefunctions. The following spectroscopic constants for 132XeH+ are obtained: Re= 1.611 ± 0.005 Å, ωe = 2313 ± 50cm-1, ωexe = 41 ± 5cm-1 and D0(Xe+ + H) = 3.90 ± 0.1 eV.
Infrared transition dipole matrix elements and probability coefficients for 132XeH+ and 132XeD+ are given. The electric dipole moment functions of the protonated rare gas atoms HeH+ to XeH+ are discussed.
The thermal and the photochemical reactions of the endoperoxide of Tetrabenzo(bc,fg.lm,pq)- pentacene have been investigated with respect to its photochromic properties. The thermal yield of irreversible rearrangement reactions was determined to be Adec = 0.045. From the activation parameters of the thermal cycloreversion. forming the parent hydrocarbon and oxygen, a half life time of 114 years at 20 °C has been extrapolated. For the quantum yield of the irreversible photochemical rearrangement a value of Qdec = 0.03 was obtained. As can be seen from the wavelength dependence of the photocycloreversion quantum yield Q1, cycloreversion occurs not only from the thermalized S2(πccπ*cc) but from higher excited states too. The maximum quantum yield was found at 302 nm to be (Q1 = 0.15. From our results it follows that this endoperoxide is of high quality in respect to the colour change colourless/blue and to thermal stability, whereas the reversibility is only moderate.
The recently developed stereospecific sodium salt glycosylation procedure has been successfully applied to the synthesis of the β-ᴅ-2′-deoxyribofuranosides of benzimidazole, 5,6-dihalogeno benzimidazoles, and some 2-substituted analogues in high yield. The 5,6-dibromo analogue was obtained by bromination of the parent nucleoside. These have all been characterized by spectroscopic methods, including 1H NMR, which permitted analyses of their solution conformations and comparison with those of the corresponding ribofuranosides. Some biological aspects, including preliminary results on cytotoxicity and antiviral activity, are briefly considered.
By means of differential thermoanalysis, the miscibility of the main polar tetraether lipid of Thermoplasma acidophilum with two ester lipids, dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol, resp., in the presence of excess water was studied. It is shown that with increasing fraction of tetraether lipid in the mixture, the transition range of dipalmitoyl phosphatidylcholine is broadened and the temperature of the maximum heat flow (Tm) is shifted to lower temperatures; furthermore, the enthaply change (ΔH) of the transition declines. Similar results were obtained with mixtures of tetraether lipid with dipalmitoyl phosphatidylglycerol. It is therefore concluded that the main polar tetraether lipid of Thermoplasma acidophilum , which essentially forms monomolecular layers, is able to form stable common phases with bilayer-forming ester lipids. Miscibility of the tetraether lipid with dipalmitoyl phosphatidylglycerol, which are both monovalent anions at neutral pH, is also observed in the presence of high proton or calcium ion concentrations.
The bipolar main tetraether lipid (MPL) of Thermoplasma acidophilum has been shown to form typical liquid expanded films at the air-water interface. The limiting molecular area at the collaps pressure is approximately Ac=73 Å2 per molecule. Monopolar aiphytanyl diether lipids were found to occupy the same area at high surface pressure as MPL. Thus, it was concluded that in the monofilm only one of the two polar headgroups of the MPL molecules is hydrated, i.e. that the single MPL molecules arc oriented upright. The packing properties of MPT. in the monofilm are determined by the properties of the branched alkyl chains only; the polar head groups do not contribute to the space requirement in the film. The collaps pressure of the MPL film is approximately 39 mN m-1 at 8°C. At a surface pressure of π = 30 mN m-1 and 20 °C the film is stable for many hours.
Potential energy, dipole moment, and electronic transition moment functions for the A 3Πand X3Σ- states of PH have been calculated from highly correlated electronic wavefunctions. The electric dipole moments in the vibrational ground state of PH are calculated to be 0.637 Debye (A 3Π) and 0.403 Debye (X3Σ-). The predicted rates of spontaneous emission between low lying vibrational states of the X state lie in the range of 46 to 109 sec-1 (PH) and 12 to 30 sec-1 (PD). The calculated radiative lifetime of the v' = 0 level in the A 3Π state of 400 ns is lower by about 10 percent than the most recent experimental value. The classical intersection of the 5Σ- and the A 3Πstate has been calculated to lie between v' = 2 and 3 with an expected uncertainty of about 500 cm−1, whereas the onset of the rotationally dependent predissociation lies at v' = 0, J' = 11.
Organodisulfide radical cations R2S2′⊕ and R2C2S2 ′⊕ can be generated from aliphatic as well as aromatic cyclic polysulfides in AlCl3/H2CCl2 solutions and characterized by their ESR spectra. Examples presented are the oxidations of 1,2,3-trithiolanes to 1.2-dithiolane radical cations, in which energetically favored planarized 3 electron/2 center bonds are formed.
Lactate dehydrogenase from pig heart is inactivated by the NAD+ -analog P1-N6-(4-azidophenylethyl)adenosine-P2-[4-(3-azidopyridinio)butyl]diphosphate (6) upon irradiation with UV light of wavelengths in the range from 300 to 380 nm. The decrease in enzyme activity can be prevented by the addition of NAD+ and oxalate. The modified enzyme shows a reduced binding capacity for its coenzyme as compared to native lactate dehydrogenase. The amount of incorporated coenzyme is deduced from the ribose content of inactivated enzyme. Tryptic digestion of the modified protein and separation of the peptides by HPLC yields 5 ribose-containing fractions. One of them, fraction 6 6 , is split by treatment with nucleotide pyrophosphatase into two subfractions, 63 and 58. Only subfraction 63 contains ribose. Whereas peptide 58 shows a UV absorption spectrum similar to that of 4-(3-aminopyridinio)-butyl phosphate (3). Amino acid analyses of the peptides indicate that the inactivator forms covalent bonds with different parts of the protein: Peptide 63 is characterized by a great portion of hydrophobic amino acids whereas peptide 58 shows a high degree of hydrophilicity.
Photofragmentation spectroscopy—the study of "half collisions" with polarized light of subdoppler line width—opens a window to look into the structure of molecules. The energy partitioning among the particular degrees of freedom of the products of the fragmentation reaction is described by the scalar properties, the direction and magnitude of a particular type of motion is described by the vector properties. The measurement of the scalar and vector properties allows a pictorial view of the intermediate state. The forces which make the fragments fly apart or rotate and vibrate can be "seen" from the line shapes. Information on the unstable intermediate state is gained from the stable fragments long after the dissociation of the parent molecule. In particular, information on the "lifetime" of the intermediate on a femtosecond time scale can be obtained.
A number of molecules, mainly three and four atomic, have been studied by this technique. Hydrogen peroxide has shown up as a textbook example. A complete analysis was possible including not only correlation of different types of fragment motion but also a correlation of the two coincident particles formed from the same parent molecule. The experimental results are in full agreement with recent calculations of the dynamics of the fragmentation on newly obtained potential energy surfaces. Hydrogen peroxide shows a strong dependence of its potential energy on the dihedral angle in the two electronic states amenable to laser excitation. This experiment further demonstrates that an analysis is also possible if two states are excited simultaneously.
Another good example is the fragmentation of hydrazoic acid for which also coincident pair correlation has been treated. Here again the results agree excellently with a qualitative picture which can be drawn from recently calculated ab initio potential energy surfaces. The HN3 example is much more complicated than the former one due to its higher structured upper potential energy surface. Strong rotational excitation is observed in the N2 fragment leaving the NH fragment rotationally cold.
The treatment of vector correlations in molecular photofragmentation is a powerful tool for the study of the dynamics of molecular dissociation reactions.
The sesquiterpenoic alcohol nerolidol was separated into its 4 stereoisomers by MPLC of the diastereomeric (1 S, 4 R)-camphanoates.
An analytical GC method was found by which both the enantiomeric pairs of (Z)- and (E)-nerolidol are resolved on a chiral cyclodextrin stationary phase. The olfactoric properties of the nerolidol stereoisomers were investigated.
By a comparative thin layer chromatographic screening of the methanol-soluble leaf exudates from more than 400 Aloe plants (183 species), 5-hydroxyaloin A was identified in 20 species. Whilst 13 of the 20 species revealed interindividual variations concerning to the occurrence of 5-hydroxyaloin A, this anthrone-C-glucosyl was unambiguously detected in each individual of 6 Aloe species. In the leaf exudates from A. marlothii Berger 5-hydroxyaloin A was only traceable in the aloin-containing chemivars. The complete anthrone-C-glucosyl pattern of these 7 clearly characterized species has been determined additionally by qualitative and quantitative high performance liquid chromatography: The results obtained demonstrate that 5-hydroxyaloin only occurs in the more stable A-configuration (10 R, 1′S), thus being till now the only anthrone-C-glycosyl which has not been found as diastereomeric pair genuinely in plants. As well, 5-hydroxyaloin A characterizes a quantitatively significant hydroxylating pathway in biosynthesis of anthranoids. It is discussed as a chemotaxonomic marker of the genus Aloe, especially of the sections Pachydendron and Eualoe.
The radiative lifetimes of the C3Il-X3II transition of the CSi radical have been calculated from highly correlated electronic wavefunctions and compared with available experimental data. For this transition, the Franck-Condon approximation fails due to the strong R-dependency of the transition moment function.
New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases
(1995)
Reactive coenzyme analogues ω-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of ω-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability at 0 °C. L actate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazonium pentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3α,20β-Hydroxysteroid dehydrogenase reacts at 0 °C with the ethyl homologue and slowly with the propyl compound. The butyl-and pentyl analogues do not inactivate at 0 °C. Tests with 14C -labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2 ′-phospho-adenosine diphosphate, a structural analogue of NADP +, was prepared by condensation of adenosine-2,3-cyclophospho-5′-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid ester with 2 ′:3′-cyclonucleotide-3′-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehyd rogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP +-dependent dehydrogenases the com pound has been show n to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.
A non-radioactive cell-free assay was developed to quantitatively determine inhibition of plant-type phytoene desaturase by bleaching herbicides. An active desaturase was prepared from an appropriately cloned E. coli transformant. Another E. coli transformant was used to produce the required phytoene. Phytofluene and t-carotene, the products of the desaturase reaction, were either determined by HPLC or optical absorption spectra. Enzyme kinetics and inhibition data for the bleaching tetrazole herbicide WL110547 are presented as an example.
To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase. KM and kcat values and the bimolecular rate constant were determined under steady- or presteady-state conditions, respectively. We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase. Mutations in this cluster do not affect the binding of cytochrome c as the KM remains largely unchanged. Rather, the kcat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer.
A detailed analysis of the chemical constituents of a Caribbean specimen of Aiolochroia crassa was performed. Five brominated products (1 -5) were isolated and one of these was a new bromotyrosine metabolite. The structure of the new compound 1 has been established from spectral studies. Compounds 1 and 2, which are the major brominated metabolites and have not been previously identified in any Aiolochroia species, could be usefully employed as chemotaxonomic markers.
Photoinduced electron transfer from organic dye molecules to semiconductor nanoparticles is the first and most important reaction step for the mechanism in the so called “wet solar cells” [1]. The time scale between the photoexcitation of the dye and the electron injection into the conduction band of the
semiconductor colloid varies from a few tens of femtoseconds to nanoseconds, depending on the specific electron transfer parameters of the system, e.g., electronic coupling or free energy values of donor and acceptor molecules [2–10]. We show that visible pump/ white light probe is a very efficient tool to investigate the electron injection reaction allowing to observe simultaneously the relaxation of the excited dye, the injection process of the electron, the cooling of the injected electron and the charge recombination reaction.
The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.
The Na+-F1F0-ATPase operon ofAcetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunita and b, a gene encoding a 16-kDa proteolipid (subunit c 1), and two genes encoding 8-kDa proteolipids (subunits c 2 andc 3). Because subunits a,b, and c 1 were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a,b, and c 1 are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunitsa, c 2, c 3,b, δ, α, γ, β, and ε. Biochemical and immunological analyses revealed that subunitsc 1, c 2, andc 3 are all part of the c-oligomer, the first of a F1F0-ATPase that contains 8- and 16-kDa proteolipids.
The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane alpha-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A(1)(-)) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp(693) to Phe resulted in an inability to photo-accumulate A(1)(-), whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp(673)) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P(700)(+) A(1)(-) radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A(1)(-) phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp(693). However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.
In der vorliegenden Arbeit werden Verfahren der Mathematik und Informatik entwickelt und eingesetzt, um Struktur, Dynamik und biologische Aktivität aus NMR spektroskopischen und empirischen Parametern zu bestimmen. Dolastatin 10 und Epothilon A sind potentielle Wirkstoffe gegen Krebs, da sie durch Wechselwirkung mit Tubulin die Zellteilung unterbinden. Die 3D Struktur beider Wirkstoffe in Lösung und die Struktur von an Tubulin gebundenem Epothilon A wird aus NMR spektroskopischen Parametern bestimmt. Dolastatin 10 liegt in einem konformationellen Gleichgewicht zwischen der cis -- und trans -- Konformation in der ungewöhnlichen Aminosäure DAP vor. Beide Konformationen des flexiblen Pentapeptids können bestimmt werden mit RMSD = 1.423 Å für das cis -- Konformer und RMSD = 1.488 Å für das trans -- Konformer. Während das trans -- Konformer gestreckt vorliegt, faltet das cis -- Konformer am DAP zurück. Epothilone A ist durch einen Makrozyklus weniger flexibel und sowohl die an Tubulin gebundene Struktur (RMSD = 0.537 Å) als auch freie Form (RMSD = 0.497 Å) kann mit geringen RMSD -- Werten bestimmt werden. Die Struktur der freien Form, welche in Lösung hauptsächlich vorliegt, ist mit der Röntgenstruktur weitgehend identisch. In der an Tubulin gebundenen Form wird eine essentielle Umorientierung der Seitenkette beobachtet, die für die Wechselwirkung mit Tubulin entscheidend ist. Dipolare Kopplungen eines Proteins sind geeignet, eine 3D Homologiesuche in der PDB durchzuführen, da die relative Orientierung von Sekundärstrukturelementen und Domänen durch sie beschrieben wird 85 . Die frühe Erkennung 3D homologer Proteinfaltungen eröffnet die Möglichkeit, die Bestimmung von Proteinstrukturen zu beschleunigen. Eine Homolgiesuche unter Nutzung dipolarer Kopplungen ist in der Lage, Proteine oder zumindest Fragmente mit ähnlicher 3D Struktur zu finden, auch wenn die Primärsequenzhomologie gering ist. Darüber hinaus wird eine Transformation für experimentelle dipolare Kopplungen entwickelt, die die indirekte Orientierungsinformation eines Vektors relativ zu einem externen Tensor in den möglichen Bereich für den Projektionswinkel zwischen zwei Vektoren und somit in eine intramolekulare Strukturinformation übersetzt. Diese Einschränkungen können in der Strukturbestimmung von Proteinen mittels Molekulardynamik genutzt werden 92 . Im Gegensatz zu allen existierenden Implementierungen wird die Konvergenz der Rechnung durch die auf diese Weise eingeführten dipolare Kopplungsinformation kaum beeinflusst. Die dipolaren Kopplungen werden trotzdem von den errechneten Strukturen erfüllt. Auch ohne die Nutzung bereits bekannter Protein oder Fragmentstrukturen kann so ein erheblicher Teil der NOE -- Information substituiert werden. Die Dynamik des Vektors, der die beiden wechselwirkenden Dipole verbindet, beeinflusst den Messwert der dipolaren Kopplung. Dadurch wird Information über die Dynamik von Molekülen auf der µsZeitskala zugänglich, die bisher nur schwer untersucht werden konnte. Die Messung dipolarer Kopplungen für einen Vektor in verschiedenen Orientierungen erlaubt die Analyse seiner Bewegung 89 . Im besonderen ist die Ableitung eines modellfreien Ordnungsparameters 2 S möglich. Weiterhin lassen sich ebenso modellfrei eine mittlere Orientierung des Vektors, axialsymmetrische Anteile und nichtaxialsymmetrische Anteile der Dynamik ableiten und auswerten. Die Anwendung der so entwickelten Protokolle auf experimentelle Daten 90 lässt Proteine deutlich dynamischer erscheinen als auf der Zeitskala der Relaxationsexperimente zu erkennen ist. Der mittlere Ordnungsparameter sinkt von 0.8 auf 0.6. Dies entspricht einer Erhöhung des Öffnungswinkels der Bewegung von ca. 22 ° auf ca. 33°. Die Bewegungen weichen teilweise bis zu 40% und im Mittel 15% von der Axialsymmetrie ab. Neuronale Netze erlauben eine schnelle (ca. 5000 chemische Verschiebungen pro Sekunde) und exakte (mittleren Abweichung von 1.6 ppm) Berechnung der 13 C NMR chemischen Verschiebung 115 . Dabei kombinieren sie die Vorteile bisher bekannter Datenbankabschätzungen (hohe Genauigkeit) und Inkrementverfahren (hohe Geschwindigkeit). Das 13 C NMR Spektrum einer organischen Verbindung stellt eine detaillierte Beschreibung seiner Struktur dar. Resultate des Strukturgenerators COCON können durch den Vergleich des experimentellen mit den berechneten 13 C NMR Spektren auf ca. 1 o/oo der vorgeschlagenen Strukturen eingeschränkt werden, die eine geringe Abweichung zum experimentellen Spektrum haben 122 . Die Kombination mit einer Substrukturanalyse erlaubt weiterhin die Erkennung wahrscheinlicher, geschlossener Ringsysteme und gibt einen Überblick über die Struktur des generierten Konstitutionssubraumes. Genetische Algorithmen können die Struktur organischer Moleküle ausgehend von derer Summenformel auf eine Übereinstimmung mit dem experimentellen 13 C NMR Spektrum optimieren. Die Konstitution von Molekülen wird dafür durch einen Vektor der Bindungszustände zwischen allen Atom -- Atom Paaren beschrieben. Selbige Vektoren sind geeignet, in einem genetischen Algorithmus als genetischer Code von Konstitutionen betrachtet zu werden. Diese Methode erlaubt die automatisierte Bestimmung der Konstitution von Molekülen mit 10 bis 20 Nichtwasserstoffatomen 123 . Symmetrische neuronale Netze können fünf bzw. sieben dimensionale, heterogene Parameterrepräsentationen der 20 proteinogenen Aminosäuren unter Erhalt der wesentlichen Information in den dreidimensionalen Raum projizieren 134 . Die niederdimensionalen Projektionen ermöglichen eine Visualisierung der Beziehungen der Aminosäuren untereinander. Die reduzierten Parameterrepräsentationen sind geeignet, als Eingabe für ein neuronales Netz zu dienen, welches die Sekundärstruktur eines Proteins mit einer Genauigkeit von 66 % im Q 3 -- Wert berechnet. Neuronale Netzte sind aufgrund ihrer flexiblen Struktur besonders geeignet, quantitative Beziehungen zwischen Struktur und Aktivität zu beschreiben, da hier hochgradig nichtlineare, komplexe Zusammenhänge vorliegen. Eine numerische Codierung der über 200 in der Literatur beschriebenen Epothilonderivate erlaubt es, Modelle zur Berechnung der Induktion der Tubulin Polymerisation (R = 0.73) und der Inhibierung des Krebszellenwachstums (R = 0.94) zu erstellen 136 . Die trainierten neuronalen Netze können in einer Sensitivitätsanalyse genutzt werden, um die Bindungsstellen des Moleküls zu identifizieren. Aus der Berechnung der Aktivität für alle Moleküle des durch die Parameter definierten Strukturraums ergeben sich Vorschläge für Epothilonderivate, die bis zu 1 000 mal aktiver als die bisher synthetisierten sein könnten.
Aging and age-related diseases are becoming more and more important for our society and our health care system. Alzheimer's disease (AD) is a disorder that destroys some parts of the brain and is characterized by global cognitive decline including a progressive irreversible loss of memory, orientation, and reasoning. “Healthy aging”, therefore, is one of the major aims for modern medicine. Apoptosis, or programmed cell death, plays an important role for example in fetal development, as well as for learning processes. T-lymphocytes usually undergo apoptosis in order to terminate an acute inflammation. The aim of this thesis was to explore the changes in the apoptotic mechanism of peripheral lymphocytes from Alzheimer’s disease (AD) patients in contrast to physiological aging. The experiments were conducted with lymphocytes of healthy volunteers of different ages, AD patients and young and aged mice. Moreover, transgenic mice carrying familiar AD-related mutations were examined. The aging study of peripheral cells of ‘healthy’-aged volunteers revealed an age-related increase of basal apoptosis. In addition, spontaneous apoptosis as well as apoptosis induced by oxidative stress (ROS) or by Fas engagement were enhanced in aging. A closer look at the subcellular basis of the lymphocytes (e.g. B-, NK-, CD4+-, and CD8+-T cells) determined that all lymphocyte subsets were affected by aging. Therefore, it could be concluded that the regulation of apoptosis is generally impaired in lymphocytes of aged persons. The increased susceptibility to oxidative stress supports the ‘Free radical theory of aging’ that claims the radicals to be the cause for the aging-process. In mice an increase of basal, spontaneous and ROS-induced apoptosis was detected in T cells from the spleen, as well. An oral treatment over two weeks with the Ginkgo biloba extract EGb761 showed a clear reduction of ROS-induced apoptosis in the treated group. Interestingly, basal and spontaneous apoptosis, e.g. physiological apoptosis, were not effected by the plant extract. This is an important benefit for therapy since physiological apoptosis has a great relevance in the elimination of cancer-cells for example. In conclusion, the antidementive drug EGb761 reduces specifically ROS-induced apoptosis that a plays an important role in aging as shown in this thesis. Based on the data found in healthy aging, lymphocytes from AD patients were assessed for apoptosis. The cells show enhanced levels of basal, spontaneous, and Fas-induced apoptosis. In subsequent experiments it was demonstrated that mainly the T cells were responsible for the findings. However, the NK-cells provided an important impact as well. In concordance with AD-affected neurons, peripheral lymphocytes of AD patients show clear signs of apoptotic cell death. In addition, basal apoptosis of T cells and the CD4/CD8-ratio showed a correlation with the severity of the dementia. Therefore, it could be speculated that apoptosis is due to activation-induced cell death (AICD) that occurs in acute and chronic activation of adaptive immunity. In AD there is a chronic neuroinflammation in the CNS triggering degeneration of neural tissue. In order to explore this, the experimental model of lymphocyte’s activation was established in healthy aging first. The study included the detection of various events of lymphocyte’s activation on the basis of the T cell subsets (CD4+ and CD8+). The inducibility to mitogenic stimulation clearly decreased in both subsets in aging. In contrast, T lymphocytes from AD patients showed an enhanced activation subsequent to mitogenic stimulation compared with age-matched nondemented persons. Only proliferation of CD8+ T cells was clearly reduced in AD. This data could be clues that an increased generation of memory T cells due to chronic neuroinflammation might be evident in AD. Memory T lymphocytes show increased inducibility upon mitogenic activation. Interestingly, CD8+ memory T cells display decreased prolifertive capacity. Due to activation, cells die by apoptosis later on. It could be concluded that AD patients display an increased amount of memory T cells compared to controls. The data implicate that there could be a cross talk between inflammatory within the brain and inflammatory cells of the periphery. This is an interesting point since the brain used to be assumed as immune-privileged zone. According to the experiment, the information of the diseased brain is transferred to white blood cells. The connection of those two compartments might raise the opportunity to observe and probably to influence easily not-accessible regions like the brain. Transgenic mice carrying mutations in familiar AD-relevant genes (Amyloid-Precursor-Protein, Presenilin-1, respectively) displayed enhanced levels of apoptotic T cells from the spleen, as well. It seems that those mutated proteins influence the regulation of apoptosis. Probably, they are involved in the increased cell death of T- and NK-cells, as well. Animals overexpressing Presenilin-1 showed reduced levels of apoptotic cell death. It was demonstrated with molecuar biology tools that Presenilin-1, processed during apoptosis, has an anti-apoptotic effect.