Effect of the Buffer Concentration on the Separation of Lactate Dehydrogenase Isoenzymes from Different Sources by Electrophoresis

Abstract

The separation of lactate dehydrogenase isoenzymes by zone electrophoresis using cellulose acetate strips as support was dependent on the concentration of the buffer used (5 mM and 50 mM, pH 7.4) and on the source of the material (chicken liver or guinea-pig liver).

In three different 5 mM buffer systems, pH 7.4 (phosphate, veronal and Tris-HC1) the four lactate dehydrogenase isoenzymes present in chicken liver cytosol: M₃H, M₂H₂, MH₃ and H₄ were resolved into four separated bands. M₃H and M₂H₂ isoenzymes migrated towards the cathode whereas the other two isoenzymes showed anodic mobilities. In 50 mM buffers, pH 7.4 all enzyme activity appeared as a single band with anodic mobility similar to that of H₄. Guinea-pig liver isoenzymes were well resolved in both buffer conditions and appeared as five bands with anodic mobilities.

The different behaviour of the lactate dehydrogenase isoenzymes in 5 mM and 50 mM buffers can not be assigned to ionic strength effects but it may explained by assuming the binding of buffer anions to the different isoenzymes. The binding would increase with the molar concentration of the buffer and reduce charge differences among the isoenzymes to different extents depending on the source of the enzyme, chicken or guinea-pig liver.     

Use of CdSe/ZnS luminescent quantum dots incorporated within sol-gel matrix for urea detection

In this work, urea detection techniques based on the pH sensitivity of CdSe/ZnS QDs were developed using three types of sol-gel membranes: a QD-entrapped membrane, urease-immobilized membrane and double layer consisting of a QD-entrapped membrane and urease-immobilized membrane. The surface morphology of the sol-gel membranes deposited on the wells in a 24-well microtiter plate was investigated. The linear detection range of urea was in the range of 0-10 mM with the three types of sol-gel membranes. The urea detection technique based on the double layer consisting of the QD-entrapped membrane and urease-immobilized membrane resulted in the highest sensitivity to urea due to the Michaelis-Menten kinetic parameters. That is, the Michaelis-Menten constant (K_m =2.0745 mM) of the free urease in the QD-entrapped membrane was about 4-fold higher than that (K_m =0.549 mM) of the immobilized urease in the urease-immobilized membrane and about 12-fold higher than that (K_m =0.1698 mM) of the immobilized urease in the double layer. The good stability of the three sol-gel membranes for urea sensing over 2 months showed that the use of sol-gel membranes immobilized with QDs or an enzyme is suitable for biomedical and environmental applications.     

Catalase adsorption onto Cibacron Blue F3GA and Fe(III)-derivatized poly(hydroxyethyl methacrylate) membranes and application to a continuous system

Poly(2-hydroxyethyl methacrylate) (poly(HEMA)) membranes were prepared by UV-initiated photopolymerization of HEMA in the presence of an initiator (a-a′-azobisisobutyronitrile, AIBN). An affinity dye, i.e. Cibacron Blue F3GA (CB) was incorporated covalently and then complexed with Fe(III) ions. The polyHEMA-CB and polyHEMA-CB-Fe(III) derivatized membranes were used in the adsorption of catalase (CAT). The enzyme-loading capability of the Fe(III)-containing membrane (23.6 μg/cm²) was greater than that of the poly(HEMA)-CB derivatized membrane (17.1 μg/cm²). The adsorption phenomena appeared to follow a typical Langmuir isotherm. The K_m values for both immobilized catalases (poly(HEMA)-CB-CAT (22.4 mM) and poly(HEMA)-CB-Fe(III)-CAT (19.3 mM)) were higher than that of free enzyme (16.5 mM). Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. A similar observation was made for the optimum pH. Operational, thermal and storage stabilities were found to increase with immobilization, especially in the presence of Fe(III). It was observed that enzyme could be repeatedly adsorbed and desorbed without significant loss in adsorption capacity or enzyme activity.     

Chitosan/N-methylol nylon 6 blend membranes for the pervaporation separation of ethanol–water mixtures

Blend membranes of chitosan and N-methylol nylon 6 were prepared by solution blending. Their pervaporation performances for the separation of ethanol–water mixtures were investigated in terms of acid (H₂SO₄) post-treatment, feed concentration, blend ratio and temperature. The pervaporation performance of the blend membranes was significantly improved by ionizing with H₂SO₄. The blend ratio of chitosan and N-methylol nylon 6 plays a different role at feed solutions of low and high water content. At a feed solution having low water content, an increase in chitosan content caused a decrease in permeability and an increase in separation factor. At a feed solution having high water content, the permeability increases with an increase in chitosan content, while the separation factor shows a maximum value around 60 wt% chitosan. It is proposed that extra permeation channels generated from the phase separation boundary between ionized chitosan and N-methylol nylon 6 account for the abnormal temperature dependence of pervaporation performance of the blend membranes.     

A lactate electrode with lactate oxidase immobilized on nylon net for blood serum samples in flow systems

Commercially available lactate oxidase from Mycobacterium smegmatis is immobilized on a nylon net which is fixed on an oxygen probe to provide a simple l-lactate sensor. A citrate buffer, pH 6.0, is the only reagent required. The high activity of the enzyme obtained with this immobilization process permits the use of only 20–100 μl of plasma; diluted with citrate buffer to 2 ml, the sample is pumped through a flow cell. The high dilution offsets inhibitory effects of some anions present in blood such as oxalate, hydrogencarbonate and chloride. The response of the sensor is linear over the range 0.2– 2 × 10^?4 M l-acetate. The lifetime is about two months. Effects of pH, temperature and different buffers are described and results on serum samples are reported.     

Continuous Lactate Measurement that Combines a Portable Ultrafiltration Storage Device with an Enzyme Sensor

ABSTRACT|A simple portable continuous L-lactate monitoring system combining elegant ultrafiltration sample collection and storage with a lactate biosensor has been developed.|The sensor has a detection range from 0.05mM to 30mM lactate dependent on different assignments of the PCS hydrogel complex layer.|The sensor shows excellent performance on sensitivity and validity as well as operational stability over one month.|Samples containing different concentrations of lactate were collected in six hours with an ultrafiltration probe and stored in a 6m long PEEK tube of 125μm in internal diameter.|The ultraslow flow rate of 100 to 350nl/min was controlled by adjusting the restriction in the portable plastic syringe pump and a constant flow can be maintained over 24 hours.|The collected samples were then detected after reversing the flow with a lactate sensor.

The total weight of the collection system is only 8g, allowing free movement of the person being tested.     

Application of screen-printed microband biosensors incorporated with cells to monitor metabolic effects of potential environmental toxins

Microband biosensors were fabricated from a screen-printed water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase or lactate oxidase enzyme. The microbiosensors were characterised for their ability to monitor ferrocyanide and H₂O₂ in phosphate buffer solution: sigmoidal cyclic voltammograms, high current density values and steady-state amperometric responses confirmed the existence of radial-diffusion-limiting microelectrode behaviour. The lactate microband biosensors were then used, in conjunction with a screen-printed Ag/AgCl reference and platinum counter electrode, to monitor lactate levels in culture medium, with a linear range of 0.5–5 mM, sensitivity of 20 nA.mM^?1, and dynamic range up to >9 mM. The lactate microband biosensors could operate continuously in culture medium over extended times (up to 24 h) at 37 °C. These biosensors were then applied to detect changes in lactate release from cultured cells in response to toxic challenge: m-dinitrobenzene (500 μM) caused a reduction in lactate production by high-passage number HepG2 single cells; D-galactosamine (20 mM) induced release of lactate by HepG2 spheroid cultures. This novel use of microband biosensors in cell culture has the potential for further application in toxicity monitoring, in both environmental and pharmaceutical areas.     

An enzyme electrode forl-lactate with a chemically-amplified response

An enzyme electrode with a chemically-amplified response forl-lactate is constructed from an oxygen electrode and a layer containing immobilized lactate oxidase, to oxidizel-lactate, and lactate dehydrogenase, to regenerate thel-lactate. Regeneration enables oxygen to be consumed beyond the stoichiometric limitation, which results in an electrode response amplified 2–250 times according to the variation in the layer properties such as the V_max and K_m values of the immobilized enzymes and the thickness of the layer. The detection limit is as low as 5 × 10^?9 M. An equation is derived to relate the rate of oxygen consumption in the layer to the experimental parameters; the equation successfully explains the experimental results.     

SYNTHESES OF SOME 35S-LABELLED ORGANIC COMPOUNDS AT BORIS KIDRIČ INSTITUTE - VINČA

Abstract

³⁵-labelled organic compounds are widely used in different fields: medicine, chemistry, biochemistry, biology, etc. Some of these compounds are known as chemoprotectors and ³⁵S-labelled forms are used in the investigation of such protective mechanism. Details of the chemical and biochemical syntheses of some ³⁵S-labelled organic compounds are given in the paper: DL-cysteine hydrochloride-³⁵S (1-amino-2-mercaptopropionic acid), vitamin B₁-³⁵S (thiamin hydrochloride), thiourea-³⁵S, carbon disulphide-³⁵S and L-methionine-³⁵S (1-amino-4-methyl- thiobutyric acid). Products of high specific activities are obtained: 5–10 mCi/mM DL-cysteine hydrochloride-³⁵S; 60–120 mCi/mM vitamin B₁-³⁵S; 100–200 mCi/mM thiourea-³⁵S; 20–100 mCi/mM carbon disulphide- ³⁵S and 1–10 Ci/mM L-methionine-³⁵S. All the products are of high chemical and radiochemical purity, which were determined by thin layer chromatography.     

Synthesis and application of a novel cold brand reactive dye to impart colouration,mosquito repellency,and UV protection to nylon

Mosquito-borne diseases are a great concern for human health, and an increment in urbanization causes a rise in the population of mosquitoes. The efficient methods and products for countering mosquito bites are the urgent need of the hour. To protect against mosquitoes, mosquito repellent textiles are an attractive substrate. An impartment of simultaneous dyeing and finishing effects on nylon is an urgent requirement as it can enhance its applicability in various technical applications. The present study reports the preparation of a mosquito repellent-cum-UV protective nylon using a novel reactive dye. The synthesis of dye involves the reaction of cyanuric chloride with H-acid (sodium 4-amino-5-hydroxy-2,7-naphthalene disulfonic acid) to generate cyanuric-H-acid, which was further reacted with diazotised 4-amino-N, N-diethyl-3-methylbenzamide (DEET-NH₂) to synthesise a novel cold brand reactive dye. Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (¹H NMR), elemental analysis (CHN analyser), UV–vis spectroscopy, and thermogravimetric analysis (TGA) were used to characterise the synthesised dye. The colouration of nylon was done in an infrared lab dyeing machine. The exhaustion and fixation of dye on nylon and the colouration properties (L*, a*, b*, C*, K/S) of dyed nylon were explored. Functional properties (mosquito repellency and UV protection) were evaluated even after laundering treatments. The dyed fabrics were also characterised using TGA and scanning electron microscope (SEM) analysis. An outstanding mosquito repellency (100 %) and good UV protection were achieved.     

New soluble polyamides with high transparence and improved gas separation properties

Abstract

Based on the aromatic diamine monomer containing di-tert-butylbenzene and methyl groups, this work proposes its polymerization with four different dicarboxylic acids. The prepared polyamides (PA 3a–3d) were characterized by GPC, FTIR, ¹H NMR, mechanical, thermal, optical and gas separated techniques. They exhibited high solubility and good optical transparency. Their optical transmittance at 450?nm wavelength was in the range of 81.4%–86.8%, and the cutoff wavelength was in the range of 327–352?nm. The membranes also had good mechanical properties with tensile strength of 79.7–91.4?MPa, elongations at breaks of 9.0–10.9% and initial modulus of 1.5–1.9?GPa. Meanwhile, these membranes possessed good thermal properties with glass transition temperature (T _g) values of 226–246?°C. The permeability of CH₄, N₂, and CO₂ for these membranes was tested by constant pressure-variable volume method. The PA 3d containing tert-butyl moiety in the diacid units exhibited highest permeability (P_CO2 = 31.4 and P_N2 = 1.9) whereas PA 3c containing hexafluoroisopropylidene moiety exhibited highest selectivity (CO₂/CH₄ = 22.2).     

A Biosensor for ADP with Internal Substrate Amplification

Abstract

An enzyme electrode was constructed from an oxygen electrode and a layer incorporating four enzyme systems for the sensitive determination of ADP and ATP. The cofactor is cycled between pyruvate kinase and hexokinase under formation of pyruvate which is detected by the coimmobilized, sequentially acting enzymes lactate dehydrogenase and lactate monooxygenase. The enzymatic recycling results in a 220-fold increased sensitivity to ADP compared to the unamplified reaction.     

Ferrocene and Ferricenium Ion as Versatile Photometric Titrants of H2O2 and D-Glucose in the Presence of Peroxidase and Glucose Oxidase. A Ferrocene-Peroxidase Stairway #

Abstract

Hydrogen peroxide can routinely be determined in the presence of ferrocene (FcH) and horseradish peroxidase by monitoring at 617 nm the enzymatically produced ferricenium dye. In contrast, D-glucose can be assayed by following the fading of the ferricenium dye FcH⁺PF₆ ^? in the presence of glucose oxidase. The change in absorbance in both cases corresponds to the amount of analyte. viz. H₂O₂ or D-glucose, in solution. The routine is very simple, invariant to the concentrations of both ferrocenes/ferricenium salt and enzyme and allows numerous “one pot” measuremeats with the detection limit of 10^?4 M for both the analytes. It takes 2–4 and 5–10 min to accomplish one analysis of H₂O₂ and D-glucose in the presence of peroxidase and glucose oxidase, respectively.

     

Electrochemical Determination of L‐Lactate Using Phenylboronic Acid Monolayer‐Modified Electrodes

The surface of a gold (Au) disk electrode was modified with a self‐assembled monomolecular layer of dithiobis(4‐butylamino‐m‐phenylboronic acid) (DTBA‐PBA) to prepare L ‐lactate‐sensitive electrodes. The DTBA‐PBA‐modified electrodes exhibited an attenuated cyclic voltammogram (CV) for the Fe(CN)₆^3? ion in the presence of L ‐lactate, as a result of the formation of phenylboronate ester of L ‐lactate accompanied with the addition of OH^? ion to the boron atom. In other words, the negatively charged DTBA‐PBA monolayer blocked the electrode surface from the access of the Fe(CN)₆^3?/4? ions. Thus, the DTBA‐PBA monolayer‐modified Au electrode can be used for determining L ‐lactate on the basis of the change in redox current of Fe(CN)₆^3?/4? ions. The calibration graph useful for determining 1–30 mM L ‐lactate was obtained.     

Flow Injection Analysis of Lactate and Lactate Dehydrogenase Using an Enzyme Membrane in Conjunction with a Modified Electrode

Abstract

A modified electrode for H₂O₂ oxidation, consisting of Pd/Au sputtered on carbon was covered with a lactate oxidase membrane and used in a FIA manifold for selective determination of lactate. The linear range was 0.01-3 mM lactate and up to 200 samples per hour were measured with a relative standard deviation of 1%. Interferences from ascorbic acid and NADH were small because of the low potential of the modified electrode. The lactate oxidase membrane electrode was also used for measurement of lactate dehydrogenase activity using direct injection of the sample into a carrier stream containing pyruvate and NAD⁺.     

Purification, Sequencing, and Biochemical Characterization of a Novel Calcium-Independent α-Amylase AmyTVE from Thermoactinomyces vulgaris

α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS–PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable α-amylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl₂, and CaCl₂, and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl₂, suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function.     

An Amperometric Glucose Sensor Based on Isoporous Crystalline Protein Membranes as Immobilization Matrix

Abstract

S-layer ultrafiltration membranes (SUMs) with an active filtration layer composed of coherent two-dimensional, isoporous protein crystals (S-layers) have been used as matrix for immobilizing monolayers of enzymes. Since S-layers are formed by periodic repetition of identical protein subunits, functional groups are present on the crystalline array in an identical position and orientation. As a consequence monolayers of enzymes can bind in a geometrically well defined way. For the covalent immobilization of enzymes carboxyl groups from the S-layer protein were activated with carbodiimide and allowed to react with amino groups of the enzyme. SUMs were employed as a new type of immobilization matrix for the developement of an amperometric glucose sensor using glucose oxidase (GOD) as the biologically active component. Glucose oxidase covalently bound to the surface of the S-layer protein retained approximately 40% of its activity. The enzyme loaded SUMs were covered with a layer of gold or platinum to function as working electrodes. These sensors yielded high signals (150nA/mm²/mmol glucose), fast response times (10–30s) and a linearity range up to 12 mM glucose. The stability under working conditions was more than 48 hours. There was no loss in activity after a storage period of 6 month.     

Influence Of Zro2 on The Physicochemical Properties of Phosphate-Based Glasses and Glass Ceramics

Abstract

Phosphate-based bioactive glasses and their glass ceramics for 47P₂O₅– (30.5)CaO–(22.5 ? x)Na₂O–xZrO₂ for different ZrO₂ contents (x = 0, 1.5, 3.0, 4.5, and 6.0 mol%) were prepared through melt quenching and controlled heat treatment procedures. The amorphous nature of glasses and the presence of crystalline phases in glass ceramics were studied by means of X-ray diffraction (XRD) studies. The density, molar volume, ultrasonic velocities, attenuation, elastic constants, and microhardness of glass and glass ceramics were used to study the structural changes. The formation of hydroxyapatite layer on the surface of glasses and glass ceramics after immersion in simulated body fluid (SBF) was explored through XRD, Fourier transform infrared (FTIR), and scanning electron microscopy (SEM) analyses. The results indicate that the added ZrO₂ increases the crosslink density of glasses, resulting in network stability, and also induces the formation of an apatite layer on the surface of glasses.

Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.     

Reagentless lactate electrodes based on electrocatalytic oxidation of flavocytochrome b2

Flavocytochrome b₂ (L-lactate :cytochrome c reductase, E.C. 1.1.2.3) from Hansenula anomala was entrapped on the surface of electrodes modified with various kinds of carbon black. The electrocatalytic oxidation of a reduced enzyme by the electroactive surface groups of carbon black enables this enzyme electrode to be used for the determination of lactate. The electrodes operate at ?0.2 to ?0.1 V vs. SCE (pH 7.0), which is low enough to avoid interference from ascorbic acid. Linear calibration graphs up to 0.5 mM lactate were obtained. Electrochemical measurements of lactate in human blood plasma and cell culture fluids showed good agreement with the results of spectrophotometric measurements.     

Biosensor Based on Self‐Assembling Glucose Oxidase and Dendrimer‐Encapsulated Pt Nanoparticles on Carbon Nanotubes for Glucose Detection

A novel amperometric glucose biosensor based on layer‐by‐layer (LbL) electrostatic adsorption of glucose oxidase (GOx) and dendrimer‐encapsulated Pt nanoparticles (Pt‐DENs) on multiwalled carbon nanotubes (CNTs) was described. Anionic GOx was immobilized on the negatively charged CNTs surface by alternatively assembling a cationic Pt‐DENs layer and an anionic GOx layer. Transmission electron microscopy images and ζ‐potentials proved the formation of layer‐by‐layer nanostructures on carboxyl‐functionalized CNTs. LbL technique provided a favorable microenvironment to keep the bioactivity of GOx and prevent enzyme molecule leakage. The excellent electrocatalytic activity of CNTs and Pt‐DENs toward H₂O₂ and special three‐dimensional structure of the enzyme electrode resulted in good characteristics such as a low detection limit of 2.5 μM, a wide linear range of 5 μM–0.65 mM, a short response time (within 5 s), and high sensitivity (30.64 μA mM^?1 cm^?2) and stability (80% remains after 30 days).