Control release of lactate dehydrogenase encapsulated in poly (vinyl alcohol) nanofibers via electrospinning

Sustained release of lactate dehydrogenase (LDH, EC 1.1.1.27) from electrospun poly (vinyl alcohol) (PVA) nanofibers was successfully achieved using the coaxial electrospinning technique. The presence of the encapsulated enzyme in the nanofibers was confirmed by infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) was used to evaluate the morphology and diameter of the nanofibers. The conversion of lactate to pyruvate by LDH coupling with the reduction of the cofactor nicotinamide adenine dinucleotide (NAD⁺) to dihydronicotinamide adenine dinucleotide (NADH) produces an increment in the ultraviolet absorption (UV) at 340 nm. This change in the UV absorbance was used to follow the release kinetic of LDH from the PVA nanofibers and also as a measure to evaluate the residual enzymatic catalytic function. Most of the encapsulated LDH enzyme was released in a sustained manner from the PVA nanofibers within a period of 1 month.     

Selective and sensitive determination of lipoyllysine (protein-bound alpha-lipoic acid) in biological specimens by high-performance liquid chromatography with fluorescence detection

The direct determination of lipoyllysine (LLys) in proteins was carried out by reversed-phase high-performance liquid chromatography with fluorescence (FL) detection. The proteins containing α-lipoic acid (LA) were first hydrolyzed with several enzymes such as pronase E and subtilisin A. The disulfide bond (-S-S-) in LLys liberated from the enzyme digestion was reduced with tris(2-carboxyethyl)phosphine to the thiol form (-SH). The reduced LLys was then labeled with ammonium 4-fluoro-2,1,3-benzoxadiazole-7-sulfonate (SBD-F) at 50 °C for 1 h. The resulting fluorophore, SBD-LLys, was separated by reversed-phase chromatography and fluorometrically detected at 510 nm (excitation at 380 nm). The calibration curve obtained from the peak areas versus the injection amounts of LLys showed a good linearity. The limits of detection and quantification of LLys on the chromatogram were approximately 0.13 pmol (signal-to-noise ratio (S/N) = 3) and 0.44 pmol (S/N = 10), respectively. A good recovery (98.9-107.1%) and precision (R.S.D.: 4.49-17.2%) of LLys were also obtained using the present procedure. The proposed method was used for the determination of LLys in spinach and animal tissues. The FL derivative was completely separated without any interference by endogenous substances in the sample and sensitively detected by the fluorimetry. The assay values of LLys per 1 g wet tissues were 3.67 μg (kidney), 1.97 μg (liver), 2.09 μg (heart), 0.59 μg (brain), 0.30 μg (lung), 0.38 μg (pancreas), and 0.20 μg (spleen). The direct determination of LLys in protein using the FL labeling method is reported for the first time.     

Molecular recognition and signal processing in biosensors

The specificity of molecular recognition is reflected to a high degree by the selectivity of the respective biosensor response. Therefore, the application of highly specific enzymes offers advantages for analytical purposes. Using lactate monooxygenase in combination with lactate dehydrogenase and pyruvate kinase, sequentially acting enzyme electrodes for lactate, pyruvate, ADP and enzyme activities, e.g. lactate dehydrogenase, creatine kinase and aminotransferases were developed. A high sensitivity was achieved based on the cycling enzyme pairs hexokinase/pyruvate kinase for ATP and ADP, and lactate monooxygenase/malate dehydrogenase for malate and oxaloacetate, respectively. On the other hand, in a sensor for a large group of substances, the unspecific microsomal cytochrome P-450 system was applied.     

486—Polarographic study of zinc bound to alcohol dehydrogenase

The differential pulse polarogram of native liver alcohol dehydrogenase consists of two peaks which correspond to the reduction of zinc associated with the enzyme. On the basis of its electrochemical properties peak I (with peak potential at ? 1.0 V) was attributed to the signal of zinc ions liberated from the enzyme adsorbed at the electrode surface. Peak II (with peak potential at ? 1.1 V) probably corresponds to the reduction of zinc still bound to the enzyme molecules adsorbed at the electrode surface. The possible difference between the contributions of catalytic and structural zinc of liver alcohol dehydrogenase to the observed currents is discussed. The polarographic behaviour of the enzyme of animal origin is compared with that of yeast alcohol dehydrogenase which yields only one peak at ? 1.0 V.The denatured alcohol dehydrogenases give a single signal at ?1.0 V which corresponds to the zinc liberated from the enzymes in the solution. The direct denaturation of liver alcohol dehydrogenase in the polarographic cell provides a quick and simple method for determination of the zinc content of the enzyme. In addition, the normal pulse polarograms of native and denatured liver alcohol dehydrogenase show considerable differences.     

Polarographic study of zinc bound to alcohol dehydrogenase

The differential pulse polarogram of native liver alcohol dehydrogenase consists of two peaks which correspond to the reduction of zinc associated with the enzyme. On the basis of its electrochemical properties, peak I (with peak potential at −1.0 V) was attributed to the signal of zinc ions liberated from the enzyme adsorbed at the electrode surface. Peak II (with peak potential at −1.1V) probably corresponds to the reduction of zinc still bound to the enzyme molecules adsorbed at the electrode surface. The possible difference between the contributions of catalytic and structural zinc of liver alcohol dehydrogenase to the observed currents is discussed. The polarographic behaviour of the enzyme of animal origin is compared with that of yeast alcohol dehydrogenase which yields only one peak at −1.0 V.The denatured alcohol dehydrogenases give a single signal at −1.0 V which corresponds to the zinc liberated from the enzymes in the solution. The direct denaturation of liver alcohol dehydrogenase in the polarographic cell provides a quick and simple method for determination of the zinc content of the enzyme. In addition, the normal pulse polarograms of native and denatured liver alcohol dehydrogenase show considerable differences.     

Immobilization of Enzymes on Polychlorotrifluoroethylene Particles Packed in Small Columns

Abstract

The use of polychlorotrifluoroethylene (PCTFE) particles packed in 5–10 cm × 0.4 cm ID columns for the immobilization of enzymes by hydrophobic adsorption is investigated. Enzyme binding capacity of PCTFE is about 0.2 mg/gm of polymer. PCTFE-immobilized lactate dehydrogenase, alcohol dehydrogenase, and urease-glutamate dehydrogenase are demonstrated to be useful for the determinations of pyruvate, ethanol, and urea, respectively. Immobilized enzyme lifetimes are generally about 1–2 months.     

Albumin coated CuInS2 quantum dots as a near-infrared fluorescent probe for NADH, and their application to an assay for pyruvate

Water-soluble CuInS₂ quantum dots (QDs) stabilized with 3-mercaptopropionic acid were synthesized in aqueous solution and then coated with bovine serum albumin. The resulting particles display fluorescence with a peak at 680 nm that is effectively quenched by 1, 4-dihydro-nicotinamide adenine dinucleotide (NADH), but not by 1, 4-nicotinamide adenine dinucleotide (NAD⁺). The enzyme lactate dehydrogenase catalyzes the reduction of pyruvate and dehydrogenation of lactic acid using NAD⁺ or NADH as a cosubstrate. The new QDs were applied to monitor the course of lactate dehydrogenase-catalyzed reaction of pyruvate by detecting NADH via its quenching effect. This resulted in a convenient and selective detection scheme for pyruvate. The detection limit is as low as 25 nM.

Determination of flunixin residues in bovine muscle tissue by liquid chromatography with UV detection.

A new and sensitive liquid chromatography-ultra violet method with a detection limit of 6 ng/g (ppb) and a limit of quantification of 15 ng/g was developed for the determination of flunixin residues in bovine muscle tissue. Flunixin in homogenized animal tissue was extracted with acetonitrile after enzyme digestion. The tissue digest (extract) was then cleaned up on a solid-phase extraction cartridge and eluted with acidified hexane. After the eluate was evaporated to dryness under nitrogen at 55 degrees C, the residue was reconstituted in 1 mL mobile phase solution and analyzed by reversed-phase gradient chromatography with UV detection at 285 nm. The method was then applied in a survey study of slaughter animals to determine whether flunixin is being used in an off-label manner for veal and beef production in Canada.     

PORPHYRIN-INDUCED PHOTODAMAGE TO ISOLATED HUMAN NEUTROPHILS

Abstract— Human neutrophils were irradiated with light at 340–380 nm in the presence of low concentrations of protoporphyrin or uroporphyrin. At increasing light doses or increasing concentrations of protoporphyrin, the neutrophils rapidly lost the ability of locomotion. Also, neutrophil chemiluminescence and hexose-monophosphate shunt activity rapidly declined. An early event was leakage of endogenous K⁺ followed by lactate dehydrogenase and at a later stage leakage of particle-bound acid phosphatase. A number of cellular enzymes were inactivated, the susceptibility to inactivation decreased in the order: succinate dehydrogenase > lactate dehydrogenase > glutamate dehydrogenase > acid phosphatase. Uroporphyrin had no effect on neutrophil functions, leakage of K⁺, or cellular enzymes. The results suggest that photodamage to the plasma membrane and the mitochondria are earlier events than photodamage to lysosomes.     

Enzymatic method for assaying calcium in serum with Ca++ -ATPase

A kinetic assay for total calcium in serum was developed which is based on the activation of Ca(++)-ATPase by free Ca(++) [Ca(++)](f) maintained by EGTA in the reaction mixture. The concentration of Ca(++)(f) was dependent on total reference calcium added or serum calcium. Ca(++)-ATPase activity was coupled to the reduction of NADH by pyruvate kinase (PK) and lactate dehydrogenase (LDH) and monitored by change in absorbance at 340 nm. The calcium in normal serum was 10.08 +/- 0.24 mg/dl (n = 35) by our method while with o-cresolphthalein complexone (CPC) method, the total calcium in the same 35 serum samples was 10.14 +/- 0.54 mg/dl. The range of within-run coefficient of variations (CVs) by this method was 0.9-2.87% at 8-12 mg/dl and day-to-day CVs were 0.72-3.17%. The presence of other ions and standard clinical interfering agents did not affect this assay system. The correlation between values obtained with our method (y) and CPC method (x) for normal serum was: y = 1.064x-0.580 mg/dl (r = 0.912, n = 59).     

Development of effective cross-linking method for bioactive substance--enzyme immobilization using glutaraldehyde oligomers

When a 10% aqueous solution of glutaraldehyde (GA) was alkalized to pH 8.5 in borate buffer solution and heated at 60 degrees C, the ultraviolet spectrum of GA solution showed two distinct absorption maxima. The one at 280 nm with a weak absorbance ascribable to the C = O bond in the aldehyde group shifted to near 300 nm after 50 min with a slight increase in its intensity. Another maximum at 235 nm with a strong absorbance was ascribable to the C = C bond of the alpha,beta-unsaturated aldehyde group which was formed by aldol condensation reaction of GA monomer, and its absorbance increased markedly with increasing reaction time. The high performance liquid chromatography (HPLC) analysis with detection at 235 nm indicated that several GA oligomers were formed by the alkali treatment and their concentrations increased. The cross-linking ability of these oligomers was examined by immobilizing enzymes (alcohol dehydrogenase (ADH), glutamate dehydrogenase (GLDH] to an aminated polymer gel matrix by reaction with the treated GA solution. The enzyme activities increased with increasing concentration of GA oligomers. Then, the GA oligomers were isolated and used as the cross-linking agent. The activities of ADH and GLDH were 4-fold and 13-fold higher, respectively, than those obtained by using untreated GA solution, while the total amounts of immobilized enzymes were almost unchanged. These results suggest that GA oligomers may act as cross-linkers in a manner different from the generally accepted Schiff base formation reaction; a possible mechanism may involve addition reaction of an amino group to the double bond in the aldol condensate of GA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of l-phenylalanine in serum by flow-injection analysis using immobilized phenylalanine dehydrogenase and fluorimetric detection

A flow-injection system with an immobilized enzyme reactor is proposed for the determination of l-phenylalanine. Phenylalanine dehydrogenase from Rhodoccus sp. M4 was immobilized on tresylated poly (vinyl alcohol) beads (13 mum) and packed into a stainless-steel column (5 cm x 4 mm i.d.). Serum sample was deproteinized with tungstic acid and filtered through an ultrafiltration membrane. The sample solution (30 mul) was injected into the carrier stream (water). The NADH formed was detected at 465 nm (excitation at 340 nm). The calibration graph was linear for 0.9-600 mum l-phenylalanine; the detection limit was 0.3 mum. The sample throughout was 25 h(-1) without carryover. The half-life period of the immobilized enzyme was 23 days.     

Flow-injection determination of 3-hydroxybutyrate in serum with an immobilized 3-hydroxybutyrate dehydrogenase reactor and fluorescence detection

A flow-injection system with an immobilized enzyme reactor is proposed for the determination of 3-hydroxybutyrate. 3-Hydroxybutyrate dehydrogenase is immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (4 cm x 4 mm I.D.). Serum is diluted and filtered. Sample solution (20 mul) is injected into the carrier stream [4mM NAD(+) in glycine buffer (pH 9.3)]. The NADH formed is detected at 465 nm (excitation at 340 nm). The calibration graph is linear for 0.7-500muM 3-hydroxybutyrate; the detection limit is 0.5muM.     

High-performance liquid chromatographic method for determining thiopental concentrations in twelve rat tissues: application to physiologic modeling of disposition of barbiturate

A sensitive, selective and reproducible high-performance liquid chromatographic assay of thiopental concentrations in twelve rat tissues was developed using thiamylal as the internal standard. Samples were homogenized in phosphate buffer, extracted into pentane and chromatographed on a microparticulate octadecyl reversed-phase column using ultraviolet detection at 290 nm. A simple digestive step with collagenase prior to homogenization facilitated analysis of thiobarbiturate in skin. Thiopental extraction recovery from fat exceeded 90%. Assay sensitivity was greater than 1 microgram/ml for tissue and plasma samples as small as 50 microliters. This assay has been applied to physiologic pharmacokinetic studies. The paper also presents typical concentration-time profiles of thiopental in four tissues taken from 74 rats given 20 mg/kg thiopental.     

High-performance liquid chromatographic determination of hydrocortisone and methylprednisolone and their hemisuccinate esters in human serum

A high-performance liquid chromatographic method is described for the simultaneous determination of methylprednisolone (MP) and methylprednisolone hemisuccinate (MPHS), or hydrocortisone (HC) and hydrocortisone hemisuccinate (HCHS) in human serum. Reversed-phase liquid chromatography was performed on a microparticulate C18 column (Spherisorb, 5 micron) using a mobile phase of 2% glacial acetic acid, 30--35% acetonitrile, 70--65% water with ultraviolet detection (254 nm). The method uses 17 alpha-hydroxyprogesterone as the internal standard for the determination of methylprednisolone and its hemisuccinate ester, or 11-deoxy-17-hydroxycorticosterone as the internal standard for the determination of hydrocortisone and its hemisuccinate ester. The sensitivity is 0.03 microgram/ml for HC, 0.07 microgram/ml for MP, 0.04 microgram/ml for MPHS, and 0.10 microgram/ml for HCHS, with a detection limit of 0.02 microgram/ml for all four steroids. Calibration curves are linear up to 3 micrograms/ml for MP or MPHS (as equivalent MP) and up to 4 micrograms/ml for HC and 7 micrograms/ml (as equivalent HC) for HCHS. The pooled relative standard deviation for replicate for each steroid is less than 7%. Plasma concentration--time curves are reported for MP and MPHS or HC and HCHS of two human subjects following intramuscular administration of 125 mg of methylprednisolone sodium succinate for injection, U.S.P., or 250 mg of hydrocortisone sodium succinate for injection, U.S.P.     

基于特异性蛋白Gαa的比浊法检测β-葡聚糖

通过基因重组技术,克隆表达了β-葡聚糖特异性结合美洲鲎G因子α亚基片段a(Gαa)蛋白,并建立了β-葡聚糖比浊检测方法。克隆的Gαa基因相对分子质量为40000(1251 bp),浓度为2 g/L,纯度达到98%以上。该蛋白能与βG特异性结合,紫外分光光度计在340 nm下检测的吸光度与βG含量呈正线性相关,线性范围3.125~200 mg/L,R2=0.997,检测限3.125 mg/L。结合力实验表明,该蛋白与βG具有良好的亲和性及特异性。该方法具有成本低、快速、专一性强等特点。     

The Pyruvate Dehydrogenase Multienzyme Complex

The three enzymes pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase constitute the pyruvate dehydrogenase multienzyme complex of E. coli; in mammals the complex also contains a kinase and a phosphatase. Multienzyme complexes are structural, functional, and regulatory units enabling the organism to operate more economically than with single enzymes. The pyruvate dehydrogenase multienzyme complex may stand at the switch-point between energy metabolism and gluconeogenesis.     

Fluorescence and quenching comparative studies of halophilic and bovine glutamate dehydrogenase

Fluorescence techniques have been used to study the structural characteristics of many proteins. The halophilic enzyme NADP-glutamate dehydrogenase from Haloferax mediterranei is found to be a hexameric enzyme composed of identical subunits. Fluorescence spectra of native and denatured halophilic and bovine glutamate dehydrogenase (h-GDH and b-GDH) have been analysed. Native h-GDH presents the maximum emission at 338 nm, whereas for b-GDH the maximum appears at 332 nm. The denaturation process is accompanied by an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum in both cases. The unfolding of h-GDH is a gradual process, which is accompanied by a loss in enzyme activity. Fluorescence quenching by external quenchers, KI and acrylamide, has also been carried out. The tryptophan residues in the protein are more exposed to the solvent in h-GDH than in b-GDH. The total amount of tryptophan residues is nearly the same for both enzymes.     

Central Carbon Metabolism in Marine Bacteria Examined with a Simplified Assay for Dehydrogenases

A simplified assay platform was developed to measure the activities of the key oxidoreductases in central carbon metabolism of various marine bacteria. Based on microplate assay, the platform was low-cost and simplified by unifying the reaction conditions of enzymes including temperature, buffers, and ionic strength. The central carbon metabolism of 16 marine bacteria, involving Pseudomonas, Exiguobacterium, Marinobacter, Citreicella, and Novosphingobium were studied. Six key oxidoreductases of central carbon metabolism, glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and isocitrate dehydrogenase were investigated by testing their activities in the pathway. High activity of malate dehydrogenase was found in Citreicella marina, and the specific activity achieved 22 U/mg in cell crude extract. The results also suggested that there was a considerable variability on key enzymes’ activities of central carbon metabolism in some strains which have close evolutionary relationship while they adapted to the requirements of the niche they (try to) occupy.     

固相萃取富集-高效液相色谱分离和测定邻甲苯胺和邻硝基甲苯

建立了以固相萃取技术进行富集 ,高效液相色谱进行分离和检测邻甲苯胺和邻硝基甲苯的方法。污染水中的邻甲苯胺和邻硝基甲苯采用Sep pakC1 8萃取柱进行固相萃取。色谱分离条件是 :Shim PackCLCODS(1 5 0mm× 4 .6mmid ,5 μm)柱为分析柱 ,甲醇 水 =60∶4 0 (V V)为流动相 ,流速为 1 .0mL min,邻甲苯胺和邻硝基甲苯的紫外检测波长分别为 2 3 0nm和 2 5 4nm ,本法具有良好的灵敏度和重现性。