Hanns-Ludwig Schmidt

Electropolymerized Azines: A New Group of Electroactive Polymers

The electropolymerization of different azines from aqueous solutions was investigated. The structure of monomers was systematically varied changing both the nature of the second heteroatom and the substituents in the aromatic rings. Considering the electropolymerization process and the properties of the resulting polymers one can denote polyazines as a new group of electroactive polymers. The electrochemical and spectroelectrochemical investigation of polyazines was done. A hypothesis on azine polymer structure is presented.

Electrocatalytic oxidation of reduced nicotinamide coenzymes at gold and platinum electrode surfaces modified with a monolayer of pyrroloquinoline quinone. Effect of Ca2+ cations

Chemisorption of cystamine and cysteamine was used for functionalization of Au and Pt electrodes, respectively with amino groups. The functionalized electrodes were used for covalent immobilization of pyrroloquinoline quinone (PQQ) as a monolayer by carbodiimide coupling of the PQQ carboxylic groups with the surface amino groups. The electrochemical properties of the PQQ-modified electrodes (formal potential E°, peak currents Ip and peak-to-peak separation ΔE) were changed after addition of Ca2+ cations, probably owing to the formation of a complex between PQQ and Ca2+. Electrocatalytic oxidation of NADH and NADPH was shown at the PQQ-modified electrodes. This process was strongly enhanced in the presence of Ca2+ cations. Cyclic voltammetry, steady-state current, rotating disk electrode and flow-injection measurements were applied to study this electrocatalytic process. The kinetic parameters of this electrocatalytic process were evaluated in the presence or absence of Ca2+ cations assuming the formation of an intermediate charge-transfer complex between the immobilized PQQ and NADH. The only influence of Ca2+ cations on the kinetics of the catalytic process is an equilibrium shift towards the formation of the charge-transfer complex [NADH ⋯ Ca2+ ⋯ PQQ]. The decay of this complex leading to the formation of the final products (NAD+ and PQQH2) was found to be independent of the presence of Ca2+ cations. The process studied is considered as a model for the mechanism of catalysis showed by PQQ- and Ca2+-containing dehydrogenases. These PQQ-modified electrodes were stable enough even in a flow-injection system and can be considered very promising for practical applications.

Systematics of 2H patterns in natural compounds and its importance for the elucidation of biosynthetic pathways

The relative global 2H-content of natural plant products is correlated to that of the primary hydrogen source, i.e. water, to the site of their biosynthesis (C3-, C4- and CAM-plants; chloroplasts, cytosol), and to their biosynthetic pathways. A relative global 2H-content sequence can be established in the order phenylpropanoids > carbohydrates > bulk material > hydrolysable lipids > steroids. A detailed analysis of the 2H-patterns of the main groups of secondary compounds reveals regularities, in that they are correlated to the primary precursors and to the origin of hydrogen from four main pools with the mean δ2H-values [‰]V−SMOW: leaf H2O ∼+30; carbohydrates ∼−70; NADPH ∼−250; flavoproteins ∼−350. Aside from the 2H-discrimination between these pools, kinetic isotope effects on defined reactions only become effective in connection with metabolic branching events. So, the 2H-pattern of natural aromatic compounds can be correlated to the 2H-pattern of the precursor carbohydrates and a reduction step in the course of the shikimic acid pathway, furthermore to the implication of the NIH-shift. The pattern of aromatic compounds from the polyketide is different from that of the shikimate pathway. The alternating 2H-abundance of fatty acid chains is caused by the origin of their hydrogen atoms from carbohydrates and from NADPH, directly or via a flavoprotein, respectively. This is similar for isoprenoids, and the natural 2H-patterns permit their assignment to the mevalonate or non-mevalonate biosynthetic pathway. Generally, the correlations and regularities of the 2H-patterns of organic compounds found are a new reliable tool for the elucidation of biosynthetic pathways and origin assignments.

Electrochemical study of pyrroloquinoline quinone covalently immobilized as a monolayer onto a cystamine-modified gold electrode

Abstract The electrochemistry of pyrroloquinoline quinone (PQQ) was studied in solubilized and immobilized states on a Au electrode modified with a chemisorbed cystamine monolayer. An electrochemically reversible diffusion-controlled reaction ( k ≈ 1.7 × 10 − 3 cm s − at pH 7.0) is observed for solubilized PQQ on the cystamine-modified electrode under acidic and neutral conditions (pH ⩽ 7) when the surface amino groups are positively charged. However, the electrochemical reduction of PQQ is completely irreversible on a non-modified Au electrode as well as on an electrode surface modified with neutral or negatively charged groups. The cystamine monolayer on the Au electrode surface was used as a basis for the covalent immobilization of PQQ via carbodiimide coupling of the PQQ carboxylic groups with the surface amino groups. The electrochemical reaction of the immobilized PQQ was reversible over a wide pH range (pH 2–11). The PQQ modified electrodes exhibited very high stability. A surface concentration of ca. 1 × 10 −10 mol cm −2 , corresponding to a monolayer, and an electron transfer rate constant k s of ca. 3.3 s −1 (pH 7.0) were evaluated for the PQQ-modified electrode. The total amount of immobilized PQQ can be increased dramatically if an Au electrode with a very high surface roughness is used. The redox potential E ° of −0.125 ± 0.003 V/SCE (pH 7.0) was obtained for both solubilized and immobilized PQQ. It is suggested that the PQQ-modified electrodes developed in this work can be used to prepare biosensors based on PQQ enzymes and to facilitate chemical reactions characteristic of PQQ itself directly on the electrode surface.

Electropolymerized Azines: Part II. In a Search of the Best Electrocatalyst of NADH Oxidation

A comparative investigation of catalytic activity of different polyazines in NADH electrooxidation is reported. The structure of azine monomers taken for electropolymerization was varied systematically changing both the second heteroatom and the substituents of aromatic rings. It was found that the monomer structure affects catalytic activity of the resulting polymer in the following way: (i) additional substitution of benzene ring by alkyl group reduced the catalytic activity, (ii) polymerized phenoxazine Brilliant Cresyl Blue was a better electrocatalyst than the corresponding phenothiazine (o-Toluidine Blue), (iii) ring substitution with only tertiary nitrogen atoms as ligands provides higher catalytic activity, (iv) higher redox potential of the polymer also provides higher catalytic activity.

Dependence of the carbon-isotope contents of breath carbon dioxide, milk, serum and rumen fermentation products on the δ13C value of food in dairy cows.

Six dairy cows of two breeds were fed during three alternating periods with products from C3- and C4- plants to yield different natural 13C enrichments of the diet (delta 13C range: -28.0 to -13.7%). The resulting changes in the 13C enrichment of breath carbon dioxide, serum and milk of the animals followed the 13C:12C of the food, in agreement with the individual biological half-lives of those products, and established isotope discriminations. Breath CO2 was more enriched in 13C than expected. This could be related to isotope discriminations during rumen fermentation. From these results an isotopic balance model for the breath CO2 could be established.

Stable isotope distribution in the major metabolites of source and sink organs of Solanum tuberosum L.: a powerful tool in the study of metabolic partitioning in intact plants

Abstract. A method was developed for the purification of main intermediates and storage products of leaves and tubers of potato for analysis of their 13C content. The method was tested for recovery of metabolites and carbon isotope discrimination during the purification process. Leaf metabolite δ13C values showed an enrichment of starch relative to sucrose and citrate. This result is in agreement with previous findings in other higher plants and indicates the existence of isotope discrimination steps during transport and metabolism of triose-phosphates in potato leaf mesophyll cells. Active anaplerotic replenishment of the tricarboxylic acid cycle in the leaves of the plants investigated was also deduced from the significant 13C enrichment of malate relative to citrate and asparagine/aspartate relative to glutamine/glutamate. Analysis of tuber metabolite δ13C values showed no difference between starch and sucrose. However, tuber sucrose appeared significantly enriched compared with leaf sucrose and also relative to tuber citrate and malate. This finding suggests the existence of sites of isotopic discrimination during sucrose processing in developing tubers. It also confirms that metabolic cycles of sucrose synthesis and breakdown and of hexose-phosphate/triose-phosphate interconversion, which have been described in excised tuber tissue, also occur in intact organs. The δ13C values were also used to estimate the metabolic rate of carbon oxidation in developing tubers on the assumption that pyruvate dehydrogenase is the main site of isotopic discrimination in the tuber cells. The result obtained was in agreement with the available literature, suggesting that analyses of natural isotopic distribution in plant products may be a useful tool for the study of metabolic processes and sink-source relationships in intact plants.

Fundamentals and systematics of the non-statistical distributions of isotopes in natural compounds

The intermolecular and intramolecular non-statistical distribution of the isotopes of the bio-elements in natural compounds must obviously be controlled by logical principles. However, a critical review of the available isotope patterns of natural compounds indicates that a previously discussed general thermodynamic order and its mechanistic foundation cannot satisfactorily explain all experimental data. In the present contribution it is shown that a partial thermodynamic order can eventually be attained for defined positions and compounds under steady-state conditions of metabolism. However, as biological systems are generally open and irreversible, many other in vivo isotope discriminations are dominated by kinetic isotope effects, even in context with reversible reactions. On the other hand, kinetic isotope effects can only become effective in vivo in combination with metabolic branching and the implied isotope shifts of the products are balanced by their relative yields. In vivo, the influences of thermodynamic and kinetic isotope effects are modulated by interferences of the actual metabolic conditions, such as the nature and kind of precursors, alternative metabolic pathways, metabolite pools and fluxes, and by reaction mechanisms. This is demonstrated by giving examples for the isotopes of hydrogen, carbon, nitrogen, oxygen and sulfur, while simultaneously the particularities of the individual elements are elaborated. The resulting general theory of the origin of non-statistical isotope distributions in biological systems permits the interpretation and prediction of isotope patterns and provides the scientific basis for the elucidation of biosyntheses and origin assignments of natural compounds.

Direct electron transfer between the covalently immobilized enzyme microperoxidase MP-11 and a cystamine-modified gold electrode

A fundamental prerequisite for the development of reagentless amperometric biosensors is the control over electron-transfer processes between immobilized biocatalytical recognition systems, e.g. enzymes, and the surface of a suitable electrode. According to Marcus’ theory [l], the electron-transfer rate is governed by the potential difference, the reorganisation energy and, most significantly for the development of enzyme electrodes, the distance between the involved redox centers. Thus, alternative electron-transfer pathways using free-diffusing redox mediators [2,3] redox-relay loaded polymers (Marcusian wires) [4,5], mediator-modified enzymes [6] and modified electrode surfaces [7,8] have been investigated aiming on a shortening of the distance between the redox sites involved in the electrontransfer reaction in question. However, the most attractive way for the development of even reagentless amperometric enzyme electrodes would be the direct communication between enzyme and electrode surface. Consequently, a relative configuration of the redox sites has to be established with the maximum distance determined according to Marcus’ theory. Especially with peroxidases, direct electron transfer has been demonstrated [9], and horseradish peroxidase has been used successfully as a biocatalytical compound for the construction of biosensors [lo]. Peroxidases adsorbed strongly at special treated carbon surfaces [ill show good electrocatalytic properties for the reduction of H,O, and have been used in combination with various oxidases for the construction of biosensors [12]. Unfortunately, the electron-transfer pathway re-

Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

The kinetic and equilibrium isotope effects on the fructose-1,6-bisphosphate aldolase reaction have been determined using the rabbit muscle enzyme. The natural 13C abundance for both atoms participating in the bond splitting were measured in position C-1 of dihydroxyacetone phosphate and glyceraldehyde 3-P after irreversible conversion to glycerol-3-P and 3-phosphoglycerate, respectively, and chemical degradation. The carbon isotope effects were determined comparing the 13C content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. 13(Vmax/Km) on C-3 was 1.016 ± 0.007 and 0.997 ± 0.009 on position C-4, and the equilibrium isotope effects K12/K13 on these positions were 1.0036 ± 0.0002 and 1.0049 ± 0.0001. The observed kinetic isotope effect on C-3 is discussed to originate from the formation of the enamine, which comes to equilibrium before the rate determining release of glyceraldehyde 3-P from the ternary complex. The equilibrium isotope effect is seen as the reason for an earlier-found relative 13C enrichment in position C-3 and C-4 of glucose and for varying enrichments in 13C of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause 13C discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.