Kinetics of immobilized pig heart fumarase

The kinetics of immobilized pig heart fumarase are described and compared with the properties of the enzyme free in solution.

1. An analogous pH dependence of initial activity is found for free and immobilized enzyme.
2. Immobilized and free fumarase deviate from classical Michaelis-Menten kinetics in the same way. The apparent K_m values are three to eight times higher for the immobilized (2 mg/g gel) enzyme.
3. The specific activity of immobilized fumarase is dependent on the final enzyme concentration on the gel; normal specific activities are observed when 50 ?g fumarase is immobilized per gram of gel, whereas the specific activity decreases with increasing enzyme concentration.
4. The activation energies for free and immobilized fumarase (50 ?g/g gel) were found to be identical between 22 and 32?C and with L-malate as substrate (E_a = 12,290 cal/mol at pH 7.9). Upon increasing the concentration of fumarase on the gel, the activation energy decreases.

Our results indicate that the true catalytic properties of fumarase are not affected by immobilization of this enzyme. The slight differences observed when fumarase is immobilized at concentrations higher than 50 ?g/g gel must be attributed to diffusional limitation at the surface of the Sepharose matrix.     

Thermoanalytical control of paint-shop wastes prior to disposal

The paint-shop wastes under study originated from metallurgical factory painting houses. Displaying a high toxicity and flammability, they are classified as specifically hazardous to the natural environment. Paint-shop wastes can be disposed of in the following ways:

solidification and deposition in sanitary landfills;

storage on a special dumping ground;

incineration.

This paper presents the physicochemical composition and thermal analysis (DTA and TG) of paint-shop wastes and their mixtures with various components. Thermoanalytical measurements were carried out in a dynamic atmosphere of air. Enthalpies were calculated from the peak areas of the DTA curves. Thermoanalytical data were compared with calorimetric results obtained with an oxygen bomb.     

Thermal analysis of some cyano compounds

The thermal behaviour of the title compounds in static air has been investigated by conventional thermal analysis (TG and DSC) and infrared spectroscopy of the solid residues at various temperatures. The following points are demonstrated:

1. the presence ofan alkali metal cation (e.g. Na(I) or K(I)) appears to be a necessary, but not sufficient condition for the intermediate formation of cyanate in the oxidation of Fe(CN) ₆ ^n? (n=3 or 4);
2. a multivalent cation favours a hydrolytic mechanism, with preferential evolution of HCN rather than H₂O;
3. the thermally induced reduction of Cu(II) to Cu(I) occurs simultaneously with the linkage isomerism of CN^?, with the resultant formation of Cu(I)-CN^? complexes.

     

Microscopic solvation: spectroscopic results vs. Monte Carlo simulations

Van-der-Waals clusters of carbazole (representing the ‘solute’) with up to 40 nitrogen or methane solvent molecules were characterized using two-color resonant two-photon ionization spectroscopy. Features in these spectra (redshift, homogeneous and heterogeneous broadening, etc.) are interpreted as being caused by various static and dynamic effects of the solvent shell surrounding the aromatic substrate. For a better understanding of such effects, Monte Carlo simulations of the clusters were performed: Statics: Using a Monte Carlo simulated annealing minimization procedure, minimum energy structures (local, probably global) were found for the various cluster species. Using a simple empirical additivity rule, spectral shifts are rationalized from these structures.Dynamics: Starting from these minimum configurations, canonical ensemble simulations were carried out in a temperature range from 0 to 35 K. Severalorder-disorder transitions were identified including:

1. orientational isomerization or ‘melting’
2. surface isomerization or decoupling
3. rigid → fluxional transitions
4. full cluster ‘melting’

We present some of our experimental results on the systems carbazole · (N₂) _n and carbazole · (CH₄) _n together with the corresponding simulation data.     

The effect of potential aqueous pollutants on the solubility of Pb+2 in cerussite—Calcite phase

The results of this work show:

1. Pb⁺² is rapidly immobilized from aqueous solutions using calcite or aragonite, CaCO₃, and the Pb⁺² is precipitated as PbCO₃ (cerussite) on the surface of the CaCO₃ (the Pb⁺²-CaCO₃ phases),
2. using CaCO₃, the concentration of Pb⁺² in certain solutions can be reduced below environmentally tolerated concentrations, and
3. organics present in solutions and natural waters can mobilize Pb⁺² from Pb⁺²-calcite phases; with effluent waters from a paper factory, and 1:1 Ca⁺²-EDTA being more effective than lignin sulfonates, 1:1 Ca⁺²-tartrate, soap, detergent, water from a sewage treatment plant, and ordinary river water.

     

CPS 758 Untersuchung des Mischungsgleichgewichts binärer Polymermischungen

The phase diagram of a binary polymer blend can be derived from the glass temperaturesT _g of the demixed phases under the following conditions:

1. The T_g's of the pure components are sufficiently different from one another.
2. TheT _g 's of the one-phase homogeneous mixtures can be determined and vary monotonically with composition.
3. The equilibrium state of the mixture is attained after sufficient long annealing at temperatureT.
4. The equilibrium state at the temperatureT can be frozen by quenching.

These conditions are examined for mixtures of polystyrene (PS) and tetra methyl-bis-phenol A-polycarbonate (TMPC). The phase separation kinetics is investigated with respect to size and composition of the co-existing phases. The co-existance curves (binodal with lower critical point) are determined for mixtures with various molar masses of the PS component. The problems concerning the cloud point curves are shortly discussed. BetweenT _g and the binodal the one-phase mixtures exist in a hindered thermodynamic state, because the crystallization of the TMPC component is a very slow process which requires very long annealing. With increasing amounts of PS in the mixture the crystallization half time of TMPC is lowered.     

SDS 800 — The new data system for quantitative depth profiling of inhomogeneous samples by SIMS

More than 13 years of SIMS application field experience of numerous users of the ATOMIKA Ionmicroprobes have been the basis for the new SIMS Data System SDS 800. The hardware and software concept of the SDS 800, therefore, pays special attention to the following requirements:

1. Convenient set-up, modification and re-use of the measuring parameter sets for easy, time-saving operation.
2. Individual parameter selection from the very broad range of SIMS measuring parameters for optimum SIMS data quality.
3. Multitasking operation for simultaneous handling of SIMS measurement, data processing, data output and of auxiliary techniques.
4. Simultaneous depth profile/ion image acquisition and processing to enhance data quality and to validate data interpretation.
5. User-friendly data processing and output.

     

Use of cyclodextrins in enzymology to enhance the solubility of hydrophobic compounds in water

In enzyme catalysis, hydrophobic compounds are usually made soluble in water by the addition of low amounts of a water-miscible organic solvent. In the present study, taking advantage of the solu-bilizing properties of inclusion compounds (cyclodextrins), a new methodology is proposed. The hydrolysis of p-nitrophenyl esters catalyzed by several hydrolases (lipase from C.cylindracea, porcine pancreatic lipase,R. arrhizus lipase and cholesterol esterase) in the presence of cyclodextrins (CD), has been investigated. This procedure presents many advantages:

1. The enzyme parameters are not affected substantially (the catalytic constant may even increase);
2. The reproducibility is higher than with previous methods;
3. CDs enhance the solubility of substrates; and
4. CDs do not destabilize enzyme structure. In some cases a thermal stabilization is observed.

     

Penetration von Polyethylenglycoldodecylethern in gespreitete Methylstearatmonoschichten

1. The penetration isotherms of octaethyleneglycoldodecyl ether and tetraethyleneglycoldodecyl ether in methyl stearate monolayers at different film areas A_M were measured at T=295 K.
2. The penetration isotherms of the system methyl stearate/octaethyleneglycoldodecyl ether depended only weakly on the area of methyl stearate. Also, the relative shift of the penetration isotherms, related to the adsorption isotherm, was small.
3. The general penetration behaviour of both the systems methyl stearate/tetraethyleneglycoldodecyl ether and methyl stearate/octaethyleneglycoldodecyl ether was nearly identical.
4. The thermodynamical analysis of the system methyl stearate/octaethyleneglycoldodecyl ether indicated that the components are only partially miscible.

     

Zustandsdiagramme binärer Phospholipidmischungen

The transition curves were calculated on the basis of two types of ideal phase diagrams:

miscibility in the liquid phase and immiscibility in the solid phase;

complete miscibility in both liquid and solid phases.

A comparison was made of the theoretical curves with observed transition curves.     

Phasenumwandlungserscheinungen in Lecithin/Wasser-Systemen

1. By means of differential-scanning-calorimetry the phase transition temperatures and -enthalpies were determined and evaluated for the three following lecithin/water systems: 1,2-dimyristoyl-lecithin/water; 1,2-dipalmitoyl-lecithin/water; 1,2-distearoyllecithin/water.
2. The preparation of the lecithin/water mixtures was made by adsorption of water from the gaseous phase. The adsorption isotherms were evaluated by the BET equation.
3. Four phase transitions were found for the monohydrates of the lecithins. The parameters depend systematically on the length of the alkyl residues.
4. In the heterogeneous two phase region the main-transition and the pre-transition occurred. The thermodynamical parameters of both transitions depend on the alkyl chain length.
5. Whole the results refer to the conclusion that the lecithin head group hydration is a stepwise process. The hydration of the first shell is finished if 5 to 6 molecules water per molecule lecithin are present, while the second hydration shell is complete when about 13 water molecules are adsorbed

     

Einfluß der radioktiven Strahlung auf das heterogene Gleichgewicht im System Festkörper-Lösung

1. The electrokinetic potentials of radioactive and nonradioactive barium sulfate samples have been determined by the streaming potential method.
2. It has been shown that depending on the method of preparation, BaSO₄ samples can have both positive and negative ξ-potentials. In the case of deposition in a neutral medium (Na₂SO₄) the BaSO₄ deposit can have a negative ξ-potential, whereas when it is prepared in an acid medium (H₂SO₄) the value of the ξ-potential is positive.
3. The introduction of radioactive sulfur-35 into BaSO₄ deposit does not affect the sign of the surface charge, but changes the electrokinetic potential value. These changes depend on the radioactive isotope content in the deposit. The extremal effect is observed with specific radioactivity 1–2 mCu/g.

     

Direct determination of pair natural orbitals

A method is proposed to solve the two-electron Schrödinger equation by a rapidly converging iterative procedure. The wavefunction is obtained in terms of its NO's. The special features of the present method are:

1. Each iteration requires only the computational equivalent of a conventional Hartree-Fock iteration.
2. Within each iteration we improve simultaneously the NO's, the CI expansion coefficients and the total energy.
3. The construction of a CI matrix is never required.

We further propose simplified NO-equations the solution of which requires a small fraction of computertime only. As examples of the efficiency of these methods we report applications to the 1¹ Sstate of He, the 1¹∑ _g ⁺ , 1³∑ _u ⁺ states of H₂, and IEPA,PNO-CI, and CEPA type computations on CH₄.     

Charakterisierung der Mischbarkeitseigenschaften binärer Monoschichten durch Messung der Gleichgewichtsspreitungsdrucke

1. The equilibrium spreading pressures were studied as a function of composition of the following binary systems by use of the Wilhelmy-method atT=295 K on aqueous substrate:

octadecyldiethylphosphine oxide/1-hexadecanol (I)

octadecyldiethylphosphine oxide/1-eicosanol (II)

octadecyldiethylphosphine oxide/stearic acid (III)

1. The equilibrium spreading pressures of the pure components of the systems I und II, respectively, decrease due to addition of the corresponding components of mixture. A minimum occurs at the mole fractionx=0,50. The equilibrium spreading pressures of mixtures of system III rise with increasing mole fraction of octadecyldiethylphosphine oxide.
2. The three spreading diagrams are based on one general type. The phase rule is applied and shows that the components of the 3 systems must be immiscible in the monolayer but miscible in the bulk phase.

     

Über die Synthese von substituierten 2-Amino-1-pyrrolinen, 1. Mitt.

Three methods for the synthesis of various substituted 2-amino-1-pyrrolines are described:

1. Cyclization of 2.2-disubstituted 4-bromobutyronitriles with ammonia to yield 3.3-disubstituted 2-amino-1-pyrrolines.
2. Reduction of 2-aminopyrrolin-5-ones or 2-iminosuccinimides with LiAlH₄, a route leading especially to 3.3.4-substituted 2-aminopyrrolines.
3. Chlorination of 4.4-disubstituted pyrrolidones with POCl₃ followed by treatment with NH₃ gives 4.4-disubstituted 2-amino-1-pyrrolines.

     

Trennung von freien und proteingebundenen Steroiden mit Amberlite

Amberlite IRA 400-coated resin strips were tested with regard to their capability to separate free from protein-bound steroids, and as such to act as an adsorbant for the determination of testosterone by protein binding methods. Results showed

1. Tritium labelled testosterone, dihydrotestosterone, cortisol, progesterone and estradiol are adsorbed to the Amberlite in aqueous media.
2. Adsorbed are mainly the free steroids and not the steroid hormones which are bound to their specific proteins.
3. Through the quantitative determination of testosterone, results show a coefficient of variation of 1.3–3.9% in the range from 1–8 ng per sample.
4. The standard curves performed on different days fit well. The percentage error calculated for curves performed on 5 successive days was 5.7% for 8 ng of testosterone and 1.7% for 2 ng.

In contrast to other adsorbants used, the Amberlite strips are much simpler to use; therefore a greater number of samples can be processed at any one time.     

Conformational preferences of the axial ligands in some metalloporphyrins. A theoretical study

The conformational preferences of the axial ligands have been determined for several metalloporphyrins MPL and MPLL′ (M = Mo, Fe; P = porphine dianion; L and L′ being the axial ligands). For MoP(C₂H₂) a qualitative analysis indicates that the conformation with the acetylenic bond eclipsing two Mo-N bonds will be favored. Ab initio SCF calculations indicate that:

1. iron porphyrins with an axial imidazole ligand show a flat potential energy curve for the rotation of the imidazole ligand;
2. iron porphyrins with a dioxygen ligand prefer the staggered conformation with the O-O bond projecting along the bisectors of the Fe-N bonds;
3. in the cis-dinitrosyl molybdenum porphyrin, the nitrosyl ligands should be eclipsed with respect to the Mo-N_pyr bonds.

These theoretical predictions are compared with the experimental structures from the literature.     

Limits of cyclodextrin application in oral drug preparations

Cyclodextrins are applied to facilitate formulation problems, to improve stability and bioavailability. Following factors are determining whether or not cyclodextrins can be applied in oral pharmaceutical preparations:

- properties of the selected CD: solubility, price, specific catalytic properties,

- the drug to be complexed: molecular weight, polarity, solubility,

- drug dose

- solubility properties of the complex and the “super solubility” /temporary over-saturation/

- complex stability and possibility to shift the dissociation equilibrium toward the appropriate direction

- legislative procedures

     

Applications of pattern recognition

This poster illustrates the lecture on Pattern Recognition and gives recently published and unpublished examples, mainly from the laboratory from the first author. The applications concern:

- the determination of metabolic pathways of branched chain fatty acids (by clustering),

- the development of a genetic classification of meteorites (by clustering),

- the classification of cholinergic agents according to their interaction with different receptors (by clustering),

- the structure of a data set consisting of gaschromatographic profiles in samples collected in pollution monitoring stations (by factor analysis and pattern recognition),

- factors determining GLC behaviour of solutes (by factor analysis and multiple regression),

- the classification of olive oils according to geographic origin (by principal components and pattern recognition),

- the diagnosis of thyroid status (by pattern recognition).

     

Recycling of NADP+ using immobilizedE. coli and glucose-6-phosphate dehydrogenase

Recycling of NADP⁺ using immobilized wholeEscherichia coli cells as source of respiratory chain, glucose-6-phosphate, and soluble yeast glucose-6-phosphate dehydrogenase (1.1.1.49) is described. NADP⁺ was recycled more than 10-fold. We demonstrated NADPH respiration at pH 5.8 inE. coli membrane vesicles. The respiratory chain was involved most probably in NADPH oxidation.

1. The respiratory activity is localized at the level of the inner bacterial membrane. The active site for NADPH facing the cytoplasm.
2. NADPH respiration is inhibited by 10 mM cyanide, similar to the conditions of inhibition of NADH respiration.
3. NADPH dehydrogenase activity seems to be the limiting step of the respiratory chain:K _M for NADPH respiration and NADPH dehydrogenase activity are similar. The pH optima for these two activities are also comparable (around pH 5.8). Furthermore, the following properties are rather in favor of a common NADH dehydrogenase and NADPH dehydrogenase activity (1.6.99.2).

o| li](1)|At saturating concentrations of NADH and NADPH, neither respiration nor dehydrogenase activities were additive. li](2)|Similar heat inactivation kinetics were observed for NADH and NADPH dehydrogenase-activity. Protection against heat inactivation was obtained for the two activities with NAD⁺, NADP⁺, NADH, and NADPH. All these results suggested the possibility of recycling of NADP⁺ under similar conditions to those previously described for NAD⁺ (Burstein et al., 1981). It becomes thus possible to use various NAD⁺ and NADP⁺-dependent dehydrogenases in enzyme technology.