Development of an amperometric biosensor based on covalent immobilization of tyrosinase on a boron-doped diamond electrode

An amperometric enzyme electrode based on direct covalent immobilization of tyrosinase on a boron-doped diamond (BDD) electrode has been developed for the detection of phenolic compounds. Combined chemical and electrochemical modifications of the BDD film with 4-nitrobenzenediazonium tetrafluoroborate, an aminophenyl-modified BDD (AP–BDD) surface was produced, and then the tyrosinase was covalently immobilized on the BDD surface via carbodiimide coupling. The response dependences of the enzyme electrode (Tyr–AP–BDD electrode) on pH of solution, applied potential, oxygen level and phenolic compounds diffusion were studied. The Tyr–AP–BDD electrode shows a linear response range of 1–200, 1–200 and 1–250 μM and sensitivity of 232.5, 636.7 and 385.8 mA M⁻¹ cm⁻² for phenol, p-cresol and 4-chlorophenol, respectively. 90 percent of the enzyme activity of the Tyr–AP–BDD electrode is retained for 5 weeks storing in 0.1 M PBS (pH 6.5) at 4 °C.     

Immobilization of Urease and Cholinesterase on the Surface of Semiconductor Transducer for the Development of Light-Addressable Potentiometric Sensors

Various methods for the immobilization of urease and butyrylcholinesterase on the insulator surface of a laser-scanned semiconductor transducer (LSST) have been tested and compared for the development of an enzyme-based light-addressable potentiometric sensor (LAPS). The method of preparing photocurable membranes on LAPS is presented, and a new type of enzyme LAPS with photocurable polymeric enzyme membranes has been elaborated. It was found that sensors prepared by means of covalent bonding and cross-linking with inactive protein (type SIII) and with photocurable membrane matrices (type SIV) are more prospective. The enzyme LAPSensors with photocurable membranes demonstrate a degree of sensitivity close to the theoretical value and working ranges of 6.3·10^–5–1.1·10^–2 and 1·10^–4–1·10^–1molL^–1 urea for acrylamide and acrylate-based membrane matrices, respectively, and 2.5·10^–4–2·10^–1molL^–1 butyrylcholine for an acrylamide membrane matrix. It is shown that such sensors can be also used for the analysis of enzyme inhibitors.     

Protease A from cotton seeds. Isolation and purification of the enzyme

Protease A has been isolated in the homogeneous state from dormant seeds of cotton plants of the Tashkent-I variety. A scheme is proposed for the isolation and purification of the enzyme which includes the following stages: extraction of the defatted seeds with 0.1 M phosphate buffer, pH 7.4; precipitation of the protein with ammonium sulfate at 60% saturation; desalting by dialysis; and ionexchange chromatography on a column containing CM- and DEAE-celluloses. The molecular weight of the enzyme has been determined as 60,000. The enzyme efficiently hydrolyzes azocasein and the 7S and 11S reserve proteins of cotton seeds. Its maximum activity appears at pH 6.4–7.4 and a temperature of 35–40°C; it is not activated by sulfhydryl reagents and loses its activity in the presence of diisopropyl phosphorofluoridate. The assumption is made that protease A belongs to the serine type of trypsin-like proteases.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 738–741, November–December, 1986.     

L-asparaginase bound in a polyacrylamide gel

Summary The enzyme L-asparaginase fromE. coli has been included in polyacrylamide gel, and some of its properties have been investigated: stability, pH dependence, heat stability, K_m. It has been shown that the enzymegel obtained has a better stability then the native enzyme.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 382–384, May–June, 1976.     

Immobilization of the enzyme L-asparaginase fromE. coli on polysaccharides IV. Covalent binding with dialdehyde-dextran

The synthesis has been effected of immobilizedE. coli L-asparaginase on medical dextran — poliglyukin. The influence of the bound polymer on some physicochemical properties of the final products have been studied. An increased resistance to heat and stability on storage of the immobilized forms of L-asparagine in comparison with a native enzyme has been found. It has been shown that the polymer modification of L-asparaginase leads to a decrease in the antigenic affinity of the immobilized enzyme as compared with the native enzyme.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga. All-Union Scientific-Research Technological Institute of Antibiotics and Enzymes for Medical Purposes, Leningrad. A. Kirkhenshtein Institute of Microbiology, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 766–771, November–December, 1986.     

Characteristics of the cholinesterase of the venom of the central Asian cobra

Summary 1. By rechromatography on sulfoethyl-Sephadex C-50, an electrophoretically homogeneous preparation of cholinesterase has been obtained from the venom ofNaja oxiana Eichwald.2. The activity of the cholinesterase isolated depends on the concentration of the enzyme and the time and temperature of incubation, and also on the pH. The following must be considered the optimum conditions: time of incubation of the enzyme with the substrate 20–30 min, pH 8.0–8.5, temperature 37–38°C.3. Diisopropyl phosphorofluoridate (DIPF) in a concentration of 2 µM completely suppresses the activity of the cobra venom cholinesterase.4. The venom cholinesterase hydrolyzes acetylcholine chloride and acetylthiocholine bromide but has no effect on butyrylthiocholine bromide, in which respect it resembles the true cholinesterases.5. Preparations of cobra venom cholinesterase do not possess a lethal action and do not potentiate the activity of the neurotoxins of the same venom.Institute of Biochemistry, Academy of Sciences of the Uzbek SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 783–789, November–December, 1972.     

Dibromine radical anion reactions with heme enzymes

Reactions of Br ₂ ^– radical anion with heme enzymes, catalase and horseradish peroxidase, have been studied by pulse radiolysis. It has been found that Br ₂ ^– does not react with the heme centre of investigated enzymes. Dibromine radical anion reacts with tryptophan residues of catalase without any influence on the activity of catalase. It is suggested that in pulse radiolysis studies, where horseradish peroxidase is at about tenfold excess towards Br ₂ ^– , the enzyme is modified rather by Br₂, than by Br ₂ ^– .     

Immunochemical characterization of a proteolytic enzyme — Protease A from cotton seeds

It has been established by double immunodiffusion in agar that protease A (a proteolytic enzyme from dormant cotton seeds hydrolyzing the native reserve proteins) is present for the first 3–4 days during the germination of the seeds. An immunological affinity between trypsin and protease A has been revealed which indicates the presence of common structural elements in them.Institute of Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 109–110, January–February, 1991.     

Chemical modification of the tryptophan residues of the L-asparaginase of E. coli with N-bromosuccinimide

Summary The role of the tryptophan residues in the L-asparaginase molecule has been studied by the method of chemical modification with N-bromosuccinimide, and it has been established that in an acid medium this reagent modifies all four tryptophan residues present in the molecule, completely suppressing the activity of the enzyme.The substrate — L-asparagine — and a competing inhibitor — S-benzyl-N-benzyloxycarbonyl-L-cysteine — protect the L-asparaginase from the action of N-bromosuccinimide, which shows the role of the tryptophan in the catalytic center of the L-asparaginase.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 228–231, March–April, 1975.     

Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles

Yeast alcohol dehydrogenase (YADH) solubilized in reverse micelles of aerosol OT (i.e., AOT or sodium bis (2-ethyl hexyl) sulfosuccinate) in isooctane has been shown to be catalytically more active than that in aqueous buffer under optimum conditions of pH, temperature, and water content in reverse micelles. Studies of the secondary structure conformational changes of the enzyme in reverse micelles have been made from circular dichroism spectroscopy. It has been seen that the conformation of YADH in reverse micelles is extremely sensitive to pH, temperature, and water content. A comparison has been made between the catalytic activity of the enzyme and the α-helix content in the conformation and it has been observed that the enzyme is most active at the maximum α-helix content. While the β-sheet content in the conformation of the entrapped enzyme was found to be dependent on the enzyme–micelle interface interaction, the α-helix and random coil conformations are governed by the degree of entrapment and the extent of rigidity provided by the micelle core to the enzyme structure.     

Protein kinase from cotton seeds

A Ca²⁺-dependent protein kinase C, active at pH 6.5–8.0 has been found in cotton seeds for the first time. The localization of the enzyme in the seeds has been established and some of its properties are described (stability in various media, capacity for performing the phosphorylation of various substrates, activation by calcium ions). Highly active preparations of cottonseed protein kinase C have been isolated by biospecific chromatography.Institute of Bioorganic Chemistry, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 255–259, March–April, 1989.     

Mimesis of peroxidase by Mn-TMPyP in the catalytic fluorescence reaction of the homovanillic acid-hydrogen peroxide system

The fluorescence produced by the catalytic effect of the manganese (III)-tetrakis-(N-methylpyridinium)porphyrin complex (Mn-TMPyP) on the oxidation of homovanillic acid by hydrogen peroxide has been studied. The reaction product fluoresces at 424 nm (with excitation at 316 nm). Traces of hydrogen peroxide (1.3 × 10^–7–2.4 × 10^–6 M) and glucose (1.5–5.0 g/ml) can be determined with good accuracy and reproducibility. The characteristics of the mimetic enzyme Mn-TMPyP have been compared with those of horseradish peroxidase.     

Physicochemical properties and substrate specificity of protease C from dormant cotton seeds

The substrate specificity of a proteolytic enzyme — protease C — isolated from cotton seeds has been studied. The activity of protease C is suppressed completely under the action of diisopropyl phosphorofluoridate. Protein inhibitors — duck ovomucoid, soybean inhibitor, and also TPCK — suppressed the activity of protease C to different degrees. On the basis of results obtained in the hydrolysis of the cottonseed reserve proteins, 7S and 11S globulins, and the B chain of insulin, protease C has been assigned to the serine group of endopeptidases. The optimum conditions — pH, time, and temperature — at which protease C exhibits its maximum activity has been determined.Institute of Chemistry of Plant Substances, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 744–746, September–October, 1988.     

Determination of the activity of neutral proteinases with respect to the homogeneous substrate 2,4-dinitrophenylglycylglycyl-L-valyl-L-arginine

Summary 1. A chromogenic specific substrate for neutral proteinases — 2,4-dinitrophenylglycylglycyl-L-valyl-L-arginine — has been synthesized.2. A method has been developed for determining the activity of neutral proteinases in enzyme preparations with the use of this substrate which is based on the spectrophotometric determination of DNP-glycylglycine formed on the cleavage of the glycyl-valyl bond in the substrate.3. The applicability of the method to the determination of thermolysin and of the neutral proteinase fromBacillus subtilis has been shown.All-Union Scientific-Research Institute of the Genetics and Breeding of Industrial Microorganisms, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 75–80, January–February, 1976.     

Enzyme sensor for L-lactate using differential pulse amperometric detection

An enzyme sensor using differential pulse (DP) amperometric detection has been developed based on the measurement of the reduction current of the oxidized form of -nicotinamide adenine dinucleotide (NAD⁺) consumed by an enzyme reaction. This biosensor has the definite advantage to prevent interference caused by electrooxidative species such as ascorbate and uric acid and exhibits higher sensitivity and selectivity in comparison to the classical DC amperometric detector. The linear detection range of this biosensor was 5.0×10^–5 — 5.0×10^–4 mol/l and the relative standard deviation (R.S.D.) at 2.5×10^–4 mol/l was 5.0%.     

Preparation of conjugates of α-amylase with a protease inhibitor and their stability

A method for obtaining conjugates of -amylase with a trypsin inhibitor and separating them into fractions has been developed. Two fractions have been obtained—thermostable and thermolabile. The thermostable fraction retained about 80% of its amylase activity after incubation at 50°C for 2 h, with activation of the enzyme during the first 30 min. In the presence of trypsin the conjugated enzyme, retained 91% of its initial activity after incubation for 1 h, although the activity of the native enzyme fell to 35% under the same conditions.Mirzo Ulugbek [Ulugh-Beg] Tashkent State University. Translated from Khimiya Prirodnykh Soedimenii, No. 2, pp. 201–204, Marhc–April, 1998.     

The lipase of the fungus Rhizopus microsporus, UZLT-1

Summary 1. An active lipase enzyme has been isolated from the culture liquid of the fungusRhizopus microsporus, UZLT-1, by precipitation with isopropanol, gel filtration on Sephadexes G-75 and G-150, and chromatography on CM-cellulose. Some properties of the purified enzyme (optimum pH, heat stability, influence of various ions) have been studied.Department of Microbiology, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 636–639, September–October, 1976.     

Isolation and study of the protease-inhibitor system of wheat grain

Summary A modified method for obtaining a protease and its natural protein inhibitor from the endosperm of wheat grain is proposed. The enzyme is purified by additional precipitation of part of the ballast proteins with ammonium sulfate, and the inhibitor by two successive precipitations of the bulk of the inactive protein with the aid of TCA of different concentrations. The specific activity of the protease increases by a factor of 5–6, and the activity of the inhibitor by a factor of 3–4. Two active isoenzymes of protease have been found in each of the varieties of wheat investigated by disk eletrophoresis.Kazakh Scientific-Research Institute of Agriculture. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 642–645, September–October, 1975.     

Affinity chromatography of pepsin using pepstatin fragments as ligands of specific sorbents

Summary 1. Specific sorbents for the affinity chromatography of pepsin on hexamethylenediamine-Sepharose using valine peptides differing by the acyl groups and configurations of the valine residue have been synthesized.2. The sorbents investigated are effective in the purification of pepsin with a low specific activity when 30–40 mg of protein is deposited on 1 ml of resin, a fourfold purification of the enzyme being achieved.M. V. Lomonosov Moscow State University. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 387–392, May–June, 1977.     

Modeling of serine protease prototype reactions with the flexible effective fragment potential quantum mechanical/molecular mechanical method

A complete cycle of chemical transformations for the serine protease prototype reaction is modeled following calculations with the flexible effective fragment quantum mechanical/molecular mechanical (QM/MM) method. The initial molecular model is based on the crystal structure of the trypsin–bovine pancreatic trypsin inhibitor complex including all atoms of the enzyme within approximately 15–18 Å of the oxygen center O of the catalytic serine residue. Several selections of the QM/MM partitioning are considered. Fractions of the side chains of the residues from the catalytic triad (serine, histidine and aspartic acid) and a central part of a model substrate around the C–N bond to be cleaved are included into the QM subsystem. The remaining part, or the MM subsystem, is represented by flexible chains of small effective fragments, whose potentials explicitly contribute to the Hamiltonian of the QM part, but the corresponding fragment–fragment interactions are described by the MM force fields. The QM/MM boundaries are extended over the C–C bonds of the peptides assigned to the QM subsystem in the enzyme, C–C and C–N bonds in model substrates. Multiple geometry optimizations have been performed by using the RHF/6-31G method in the QM part and OPLSAA or AMBER sets of MM parameters, resulting in a series of stationary points on the complex potential-energy surfaces. All structures generally accepted for the serine protease catalytic cycle have been located. Energies at the stationary points found have been recomputed at the MP2/6-31+G^* level for the QM part in the protein environment. Structural changes along the reaction path are analyzed with special attention to hydrogen-bonding networks. In the case of a model substrate selected as a short peptide CH₃(NHCO-CH₂)₂ – HN–CO–(CH₂–NHCO)CH₃ the computed energy profile for the acylation step shows too high activation energy barriers. The energetics of this rate-limiting step is considerably improved, if more realistic model for the substrate is considered, following the motifs of the ThrI11–GlyI12–ProI13-–CysI14–LysI15–AlaI16–ArgI17–IleI18–IleI19 sequence of the bovine pancreatic trypsin inhibitor.