Regularities of Bonding of Chlorin e 6 to the Oligomeric Enzyme Lactate Dehydrogenase

Using differential-spectrophotometry, spectral-luminescence, and polarization methods, we have investigated regularities of complexing of a promising photodynamic sensitizer — chlorin e ₆ — with a key glycolytic enzyme — lactate dehydrogenase (LDH). The parameters of the dye–enzyme complex have been estimated by the difference between the spectral characteristics of the free dye and the dye bonded to the enzyme. It is shown that the tetrameric LDH molecule forms an equilibrium complex with four chlorin molecules and the sensitizer is bonded independently to each subunit entering into the composition of the tetramer. It has been established that the spectral characteristics of chlorin bonded to LDH are sensitive to the structure transformations arising in the active center of the enzyme as a result of the formation of an unproductive enzyme–coenzyme–substrate complex, which allows the conclusion that the dye is localized in the neighborhood of the active center of LDH.     

Photomodulation of glutamate dehydrogenase properties by red light

To gain some insight into the mechanism by which red light-biosystem interaction occurs, an investigation was made of certain features of purified glutamate dehydrogenase from beef liver (E.C. 1.4.1.3.) irradiated with either an He---Ne laser (632.8 nm) or a red light-emitting diode (650±20 nm). In both cases the energy dose was 0.24 J cm⁻². Significant changes in the glutamate dehydrogenase extinction coefficient measured at 275 nm, the capability of the enzyme to bind the reduced form of nicotinamide adenine dinucleotide (NADH), certain kinetic parameters, the pH and temperature dependence and the sensitivity to guanosine 5 triphosphate (GTP) and adenosine diphosphate (ADP) were found, probably due to the interaction of lightwith a protein domain containing a metal ion or ions. He---Ne laser and diode irradiation were found to differ with regard to their interaction with glutamate dehydrogenase. Interestingly, different effects were also found when an He---Ne laser and a non-coherent Xe---Hg lamp were used to irradiate glutamate dehydrogenase under the same experimental conditions. This confirms that non-coherent light at various power levels affects the isolated glutamate dehydrogenase.     

乳酸脱氢酶反应器用于流动注射分析测定人体液中的L-乳酸

本文用[3-(2-氨乙基)氨丙基]三乙氧基硅烷试剂胺化一定孔径大小的载体,用戊二醛作偶联剂,将乳酸脱氢酶固定于其上,装入柱内,联结于流动体系中。L-乳酸的浓度在0.1～4.4mmol/L呈线性,检测限为0.4nmol(s/N=4),相对标准偏差≤1.2％,回收率在96％～103％。每小时进样35次。测定了固定化LDH的表观米氏常数及人血清和尿中的L-乳酸。     

Polyelectrolyte complex capsules as a material for enzyme immobilization

The polyelectrolyte complex (PEC) membrane formed by cellulose sulfate and poly(dimethyldiallylammonium chloride) was used to encapsulate lactate dehydrogenase. The exclusion limit of the membrane was found to be low enough to secure irreversible entrapping of the enzyme. The obtained capsules were checked for their functionality in a stirred-batch reactor by following the kinetics of NADH oxidation. The data were fitted with an isotropic kinetic model including competitive product-inhibition phenomenon. The results of mathematical modeling demonstrated that the anisotropic system, like PEC capsules, could be satisfactorily described by the isotropic model.     

A Biomimetic Nickel Complex with a Reduced CO2 Ligand Generated by Formate Deprotonation and Its Behaviour towards CO2

Reduced CO₂ species are key intermediates in a variety of natural and synthetic processes. In the majority of systems, however, they elude isolation or characterisation owing to high reactivity or limited accessibility (heterogeneous systems), and their formulations thus often remain uncertain or are based on calculations only. We herein report on a Ni?CO₂^2? complex that is unique in many ways. While its structural and electronic features help understand the CO₂‐bound state in Ni,Fe carbon monoxide dehydrogenases, its reactivity sheds light on how CO₂ can be converted into CO/CO₃^2? by nickel complexes. In addition, the complex was generated by a rare example of formate β‐deprotonation, a mechanistic step relevant to the nickel‐catalysed conversion of H_xCO_y^z? at electrodes and formate oxidation in formate dehydrogenases.     

Creating a Low‐Potential Redox Polymer for Efficient Electroenzymatic CO2 Reduction

Increasing greenhouse gas emissions have resulted in greater motivation to find novel carbon dioxide (CO₂) reduction technologies, where the reduction of CO₂ to valuable chemical commodities is desirable. Molybdenum‐dependent formate dehydrogenase (Mo‐FDH) from Escherichia coli is a metalloenzyme that is able to interconvert formate and CO₂. We describe a low‐potential redox polymer, synthesized by a facile method, that contains cobaltocene (grafted to poly(allylamine), Cc‐PAA) to simultaneously mediate electrons to Mo‐FDH and immobilize Mo‐FDH at the surface of a carbon electrode. The resulting bioelectrode reduces CO₂ to formate with a high Faradaic efficiency of 99±5 % at a mild applied potential of ?0.66 V vs. SHE.     

Immobilization and Characterization of Lactate Dehydrogenase on TEOS Derived Sol-Gel Films

Physical adsorption and physical entrapment techniques have been utilized for the immobilization of lactate dehydrogenase (LDH) on tetraethylorthosilicate (TEOS) derived sol-gel films. The enzyme (LDH) activity has been assayed as a function of time, temperature, pH and pyruvate concentration. The results of photometric measurements used for monitoring the reaction yield a response time of about 1 min, linearity over a concentration range of 0–1.5 × 10^-3 M and detection limit of 5 × 10^-5 M. The TEOS sol-gel films containing LDH have been found to be stable for about 30 days at temperatures 4 to 10°C.     

Flow injection determination of histamine with a histamine dehydrogenase-based electrode

A flow injection analysis (FIA) system for the determination of histamine was developed using histamine dehydrogenase (HmDH)-based electrode. Histamine dehydrogenase was immobilized in an osmium-derivatized redox polymer, poly(1-vinylimidazole) complexed with Os(4,4′-dimethylbipyridine)₂Cl₂ (PVI-dmeOs), film on a glassy carbon electrode. As expected from the characteristics of this enzyme in a solution, this electrode exhibits high selectivity to histamine and is not sensitive to other primary amines including common biogenic amines, putrescine, cadaverine and tyramine. The detection limit for histamine was 100 pmol ( μl injection) at a S/N ratio of 3, and response linearity was retained up to 0.6 mM. The FIA system was successfully applied to the determination of histamine in fish samples. The performance of the FIA system is discussed and compared with a high-performance liquid chromatography (HPLC) method which is routinely used for histamine analysis.     

Selection and microencapsulation of an “NADH-oxidizing” bacterium and its use for NAD regeneration

An alternative approach to the regeneration of coenzymes is described here using immobilized microorganisms possessing “NADH-oxidase” function. Bacteria containing NADH-oxidase activity are immobilized by microencapsulation within artificial cells. In this form, the microencapsulated bacteria can recycle NADH back to NAD in the presence of molecular oxygen as an electron acceptor. The only byproduct of the recycling reaction is water. In order to perform the biological regeneration of NAD, the activity of NADH-oxidase was investigated in 13 strains of aerobic bacteria and yeast. The NADH-oxidizing bacteriaLeuconostoc mesenteroides exhibited the highest activity among the microorganisms tested. The permeabilized bacteria showed 10% of their initial activity after microencapsulation. Light and electron microscopy studies of bacteria loaded microcapsules have been done. Enzymatic properties of microcapsule-immobilized bacteria were investigated in comparison with those of the free enzyme complex.Leuconostoc mesenteroides, containing NADH-oxidase, has been microencapsulated together with 3α-hydroxysteroid dehydrogenase (3α-HSDH) for stereospecific steroid oxidation. In a batch reactor, 2 mg of NAD, with recycling, allowed the same substrate consumption as 4.4 mg of NAD without recycling. The microencapsulated system can be used repeatedly. The system is functional for 10 h, during which time each molecule of NAD has been used 7.6 times.     

Effect of pH on the stability of hexokinase and glucose 6-phosphate dehydrogenase

Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical methods. The stability of the enzymes can be affected by several variables, pH being one of them. The effect of pH on the stability of HK and G6PDH was evaluated in this work. Baker’s yeast cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 5.0 mM MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors. The cell-free extract was obtained by centrifugation (2880g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5). The residual activity of HK and G6PDH, expressed as μmol of NADPH formed per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4°C. It was observed that up to 4 h both enzymes were stable in all buffers used. However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at pH 7.0, 9.5, and between 4.5 and 5.5.