Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.     

Isolation,purification and characterization of pepsin soluble collagen isolated from silvertip shark (Carcharhinus albimarginatus) skeletal and head bone

Type II pepsin soluble collagens (PSC) were isolated from skeletal and head bone of silvertip shark; and examined for their biochemical and structural properties. Among the raw materials, the protein content (8.99%) was high in skeletal bone and the ash content (28%) was high in head bone. After the collagen extraction, the raw materials contained higher amount of ash content ranging from 82 to 88%. The hydroxyproline content of skeletal and skeletal PSC (30 and 113 mg/g) was higher than those head and head PSC. Both collagens were composed of two different α-chains (α₁- and α₂-chains) and were characterized as type II collagen. Amino acid analysis of skeletal and head PSC indicated imino acid contents of 156 and 175 amino acid residues per 1000 residues, respectively. Similar, Fourier transform infrared spectra of SCII and HCII were observed, which suggested that the isolation process did not affect the secondary structure and molecular order of collagen, particularly the triple–helical structure. Denaturation temperature of skeletal PSC (31 °C) was higher than that of head PSC. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. These results suggested that the PSC isolated from skeletal and head bone of silvertip shark were found to be suitable biomaterial in commercial applications as alternatives to mammalian collagen.     

Structure and composition of teleost scales from snakehead Channa argus (Cantor) (Perciformes: Channidae)

The structure of teleost scales from snakehead Channa argus was investigated using thermogravimetric analysis (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive analysis of X-rays (EDAX), Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction (XRD). Thermal treatment of fish scales indicates that the fibrillary plate is partially calcified. SEM shows two kinds of scale denticles, arranged along the circuli in the anterior field and the lateral fields, respectively. TEM indicates the stratum laxum with abundant fibrils, chromatophores and capillary blood vessels within the scale covering, and shows the fibrillary plate as an 'orthogonal plywood structure' of stratified lamellae, consisting of 80–100 nm diameter collagen fibres co-aligned in individual lamellae and alternated by c. 90° of the fibre alignment between adjacent lamellae. EDAX, FTIR and XRD show that the mineral phase of the scales is a carbonated hydroxyapatite with a Ca:P molar ratio of 1·85.     

Development of an effective process for utilization of collagen from livestock and fish waste

A procedure for the extraction of protein and production of peptides by enzymic hydrolysis from bone and skin wastes containing collagen was developed. Fat and inorganic components were first removed in a pretreatment step and a high molecular weight protein extracted under acidic conditions (pH 3) using a 1 h reaction time at 60 °C. The molecular weight of extract from pig skin was greater than 100 kDa. The extract had a high water retention capacity, was beneficial for repair of rough skin, had no odor problem and was demonstrated to be safe in skin patch tests. It was thus considered acceptable for use in cosmetic materials. Pretreated fish bone and pig skin were hydrolyzed with a commercial enzyme. The hydrolysates had a high anti-radical activity (IPOX₅₀, 0.18 and 0.45 mg ml⁻¹) and a high potential for decreasing blood pressure (IC₅₀, 0.16 and 0.41 mg ml⁻¹), suggesting the hydrolysates could be a useful additive in food materials.     

Isolation and characterization of thermostable collagen from the marine eel-fish (Evenchelys macrura)

Aim and methodsCollagen is the most abundant protein found in animal body, which is widely used for biomedical and pharmaceutical applications. In the present study, acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin wastes of marine eel fish (Evenchelys macrura) were isolated and characterized.ResultsASC and PSC extracted from eel fish skin showed the yields of 80 and 7.10 percent (based on dry weight), respectively. ASC and PSC comprising different α-chains (α1, α2 and α3) were characterized as type I and exhibited high solubility in acidic pH (1–4) and were soluble in the presence of NaCl at concentration up to 3.0 and 4.0 percent (w/v) for ASC and PSC, respectively. Amino acids analysis of both ASC and PSC contained imino acid of 190 and 200 residues per 1000 residues, respectively. The present results of ASC and PSC from eel fish skin exhibited higher thermal stability of 39 °C and 35 °C, respectively. Similar, Fourier transform infrared (FTIR) spectra of ASC and PSC were observed and suggesting that pepsin hydrolysis did not affect the secondary structure of collagen, especially triple-helical structure.ConclusionThese results suggest that the marine eel fish skin collagen close to the T_d (denaturation temperature) of mammalian collagen which could be used in the biomedical materials, food and pharmaceutical industries as an alternative source.     

The complete mitogenome of the snakehead Channa argus (Perciformes: Channoidei): genome characterization and phylogenetic implications

To better understand the phylogenetic status of the snakehead, Channa argus, we determined its complete mitogenome sequence using long-polymerase chain reaction and the direct sequencing method. The complete mitogenome sequence was 16,559?bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region (D-loop), the gene composition/order of which was identical to that observed in most other vertebrates. This was the first report of the mitogenome sequence in suborder Channoidei. Phylogenetic relationships of 14 perciform suborders based on mitogenome sequences were reconstructed using Bayesian inference and maximum likelihood methods. The results strongly supported the monophyly of Perciformes and the snakehead, as a representative species of suborder Channoidei, formed the most basal branch having sister relationship with the clade containing all other analyzed perciform fishes. The further phylogenetic analyses of six channid species, based on cytochrome b gene, suggested that two channid genera constituted reciprocally monophyletic clades. In addition, the relaxed molecular clock method was used to estimate divergence dates among major suborders of Perciformes and major species in Channoidei.     

Thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) from Lenzites betulinus

A thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) was purified from the culture medium of Lenzites betulinus by ion exchange chromatography, gel filtration and isoelectric focusing chromatography. The MnP purified from L. betulinus (L-MnP) has a molecular mass of 40 kDa and its isoelectric point was determined to be 6.2. The first 19 amino acids at the N-terminal end of the L-MnP sequence were found to exhibit 74% identity with those of a Phlebia radiata MnP. L-MnP was proved to have the highest hydrogen peroxide tolerance among MnPs reported so far. It retained more than 60% of the initial activity after thermal treatment at 60°C for 60 min, and also retained more than 60% of the initial activity after exposure to 10 mM hydrogen peroxide for 5 min at 37°C.     

Extraction and characterization of gelatin from camel skin (potential halal gelatin) and production of gelatin nanoparticles

Gelatin is used as an ingredient in both food and non-food industries as a gelling agent, stabilizer, thickener, emulsifier, and film former. Porcine skins, bovine hides, and cattle bones are the most common sources of gelatin. However, mammalian gelatins are rejected by some consumers due to social, cultural, religious, or health-related concerns. In the present study, gelatin was obtained from camel skin as an alternative source using a combination of processing steps. Central composite design combined with response surface methodology was used to achieve high gelatin yields under different extraction conditions: temperatures of 40, 60, and 80 °C; pH values of 1, 4, and 7; and extraction times of 0.5, 2.0, and 3.5 min. Maximum gelatin yield from camel skin (29.1%) was achieved at 71.87 °C and pH 5.26 after 2.58 min. The extracted gelatin samples were characterized for amino acid profile, foaming capacity, film formation, foam stability, and gel strength (Bloom value). Gelatin nanoparticles were produced, and their morphology and zeta potential were determined. Bloom value of the camel skin gelatin was 340 g. Amino acid analysis revealed that the extracted gelatin showed high glycine and proline contents. Analysis of camel skin gelatin nanoparticle and functional properties revealed high suitability for food and non-food applications, with potential use in the growing global halal food market.     

Electrochemical measurement of hydrogen peroxide in plasma: evaluation of freshwater prawn (Macrobrachium rosenbergii) and crucian carp (Cyprinus carpio) phagocytes under natural conditions

An electrochemical technique for the real-time detection of hydrogen peroxide (H2O2) was employed to describe respiratory burst activity (RBA) of phagocytes in plasma which can be used to evaluate the ability of immune system and disease resistance. The method is based upon the electric current changes, by redox reaction on platinum electrode of extracellular hydrogen peroxide (H2O2) released from phagocytes stimulated by the zymosan at 680 mV direct current (d.c.). Compared with the control, activation of respiratory burst by zymosan particles results in a high amperometric response, and a current peak was obtained during the whole monitoring process. The peak current was proved by addition of Cu2+ and other controls, to be the result of intense release of H2O2 from phagocytes. The peak area was calculated and used to evaluate the quantity of effective H2O2, which represents the quantity of H2O2 beyond the clearance of related enzymes in plasma. According to Faraday's law, the phagocytes' ability of prawns to generate effective H2O2 was evaluated from 1.253 x 10(-14) mol/cell to 6.146 x 10(-14) mol/cell, and carp from 1.689 x 10(-15) mol/cell to 7.873 x 10(-15) mol/cell. This method is an acute and quick detection of extracellular effective H2O2 in plasma and reflects the capacity of phagocytes under natural conditions, which could be applied for selecting species and parents with high immunity for breeding in aquaculture.     

Reaction of iron(III)-polyaminocarboxylate complexes with hydrogen peroxide: Correlation between ligand structure and reactivity

Reactions of iron(III) complexes with five polyaminocarboxylates and hydrogen peroxide in an alkaline solution were investigated. Iron(III) complexes of which the ring including two nitrogen and iron atoms is five-membered formed a well-known stable side-on peroxo adduct. On the other hand, iron(III) complexes which have a six-membered ring formed a short-lived side-on peroxo adduct and then changed to iron(II) complex and superoxide. Electrochemical measurements showed that the redox potentials of the iron complexes having a six-membered ring are higher than those of the complexes having a five-membered ring. These results indicate that the chelate size is an important factor for tuning the redox potential of the iron center and for the reactivity toward hydrogen peroxide.     

Purification and characterization of two pepsins from the stomach of pectoral rattail (Coryphaenoides pectoralis)

Two pepsins (A and B) were purified from the stomach of pectoral rattail (Coryphaenoides pectoralis) by acidification, ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography to obtain a single band on native-PAGE and SDS-PAGE. The purities of pepsin A and B were increased to 7.1- and 13.0-fold with approximately 5.7% and 2.2% yield, respectively. Pepsin A and B had the apparent molecular weights of 35 and 31 kDa, respectively, when analyzed using SDS-PAGE and Sephacryl S-200 gel filtration. Pepsin A and B showed maximal activity at pH 3.0 and 3.5, respectively, and had the same optimal temperature at 45 °C using hemoglobin as a substrate. Both pepsin A and B were stable in the pH range of 2.0–6.0 but were unstable at the temperatures greater than 40 °C. Activity of both pepsins was inhibited by pepstatin A and was activated by divalent cations, indicating pepsin characteristics. Activities of both pepsins continuously decreased as NaCl concentration increased (0–30%). The enzymes had high affinity and activity toward hemoglobin with K_m and K_cat values of 98–152 μM and 32–50 S^− 1, respectively. Purified pepsins generally showed the similar characteristics to other fish pepsins.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Mechanical properties of pufferfish (Lagocephalus gloveri) skin and its collagen arrangement

Skin is a biological material the mechanical properties of which are dependent on the constituents from which it is assembled. Skin, the outer covering of animals is made up of collagen fibers arranged in more or less ordered arrays. Pufferfish skin provides a rigid framework to support the body contents and a flexible covering to allow whatever changes are necessary for the remarkable inflation mechanism. Here, we describe the structure and tensile properties of the dorsal and ventral skin of the pufferfish, Lagocephalus gloveri Abe and Tabeta, 1983. The ultimate tensile strength of ventral skin was found to be around two times higher than that of the dorsal skin. It was observed that the dorsal skin could resist more deformation than the ventral skin. The collagen fibers were arranged in different ordered arrays for ventral and dorsal skin and the concentration of fibers was found to be more in ventral than dorsal skin. This provides more stiffness to ventral skin. Scanning electron microscopy studies of the ventral skin showed a unidirectional arrangement of the collagen fibers, which provides more stretching capacity. Dorsal skin, on the other hand, has an orthogonal arrangement of fibers. The present study thus showed that the mechanical behavior of the skin of L. gloveri is strongly influenced by the concentration and arrangement of collagen fibers.     

The kinetics of the complex formation between iron(III)-ethylenediaminetetraacetate and hydrogen peroxide in aqueous solution

The kinetics of the formation of the purple complex [Fe^III(EDTA)O₂]³⁻, between Fe^III-EDTA and hydrogen peroxide was studied as a function of pH (8.22-11.44) and temperature (10-40 °C) in aqueous solutions using a stopped-flow method. The reaction was first-order with respect to both reactants. The observed second-order rate constants decrease with an increase in pH and appear to be related to deprotonation of Fe^III-EDTA ([Fe(EDTA)H₂O]⁻ ⇔ Fe(EDTA)OH]²⁻ + H⁺). The rate law for the formation of the complex was found to be d[Fe^IIIEDTAO₂]³⁻/dt=[(k₄[H⁺]/([H⁺] + K₁)][Fe^III-EDTA][H₂O₂], where k₄=8.15±0.05×10⁴ M⁻¹ s⁻¹ and pK₁=7.3. The steps involved in the formation of [Fe(EDTA)O₂]³⁻ are briefly discussed.     

Reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations using synthetic binuclear Mn(II) and Mn(IV) complexes: production of hydrogen peroxide

Reconstitution of Mn-depleted PSII particles with synthetic binuclear Mn complexes (one Mn(II)₂ complex and one Mn(IV)₂ complex) was examined. In both cases the electron-transfer rates in the reconstituted systems were found to be up to 75–82% of that measured in native PSII but the oxygen evolution activity remained lower (<5–40%). However, hydrogen peroxide was also produced by the reconstituted samples. These samples therefore represent a new type of reconstituted PSII that generates hydrogen peroxide as the final product in reconstituted PSII centers.     

Structural studies on manganese(III) and manganese(IV) complexes of tetrachlorocatechol and the catalytic reduction of dioxygen to hydrogen peroxide

The mononuclear complexes (Bu₄N)[Mn(Cl₄Cat)₂(H₂O)(EtOH)] and (Bu₄N)₂[Mn(Cl₄Cat)₃] (H₂Cat=1,2-dihydroxybenzene) have been synthesised and characterised by X-ray diffraction. This work provides a direct, independent, synthesis of these complexes and an interesting example of how solvent effects can promote the formation of either a manganese(III) or manganese(IV) complex of the same ligand. The characterisation of (Bu₄N)[Mn(Cl₄Cat)₂(H₂O)(EtOH)] supports previous work that manganese(III) is extremely reluctant to form tris (catecholato) complexes due to the short `bite distance' of catecholate oxygen atoms (2.79 Å) which are unable to span the elongated coordination axes of the Jahn-Teller distorted Mn(III) ion and explains the 2:1 and 3:1 tetrachlorocatechol:manganese ratios in the Mn(III) and Mn(IV) complexes, respectively. Hydrogen peroxide production using dioxygen and hydroxylamine as substrates in acetonitrile/water mixtures, under ambient conditions, can be demonstrated with both complexes, suggesting that neither labile coordination sites nor the oxidation state of the manganese are important to the catalytic system. Turn over frequencies (TOF, moles of H₂O₂ per moles of manganese per hour) of ∼10 000 h⁻¹ are obtained and this compares very favourably with the commercial production of hydrogen peroxide by the autoxidation of 2-ethylanthrahydroquinone (AO process).     

On the interaction of compounds of chromium(VI) with hydrogen peroxide. A study of chromium(VI) and (V) peroxides in the acid-basic pH range

A computational study of chromium(VI) and (V) peroxides, which exhibit important genotoxic and mutagenic activity, is reported. Energies and equilibrium geometries for [Cr^VI(O)(O₂)₂(OH)]⁻, [Cr^VI(O)(O₂)₂(OH₂)], [Cr^VI(O)(O₂)₂(py)], [Cr^VI(OH)(O₂)₂(OH₂)]⁺, [Cr^V(O)(O₂)₂(OH₂)]⁻ and species were calculated using molecular mechanics calculations (MMFF94 and MM⁺), quantum calculations with semi-empirical methods (RHF and UHF/PM3) and density functional theory (pBP86/DN* or pBP/DN* and B3LYP/6-31G(d). Equilibrium geometries for the compounds [Cr^V(O₂)₃(OH)]²⁻ and [Cr^V(O₂)₄]³⁻ were determined by molecular mechanics. Vibrational frequencies, standard thermodynamic quantities and electronic spectra were calculated using B3LYP/6-31G(d). The structural relationship between all these species and an explanation of the formation of peroxo species in the acid-basic pH range are given. An experimental study of peroxo species in basic medium was also performed (synthesis, X-ray powder diffraction patterns and infrared spectra of the peroxo complexes isolated) but did not confirm the existence of a tri-peroxo complex in the solid phase.     

Tryparedoxin peroxidases from Trypanosoma cruzi: high efficiency in the catalytic elimination of hydrogen peroxide and peroxynitrite

During host cell infection, Trypanosoma cruzi parasites are exposed to reactive oxygen and nitrogen species. As part of their antioxidant defense systems, they express two tryparedoxin peroxidases (TXNPx), thiol-dependent peroxidases members of the peroxiredoxin family. In this work, we report a kinetic characterization of cytosolic (c-TXNPx) and mitochondrial (m-TXNPx) tryparedoxin peroxidases from T. cruzi. Both c-TXNPx and m-TXNPx rapidly reduced hydrogen peroxide (k = 3.0 × 10⁷ and 6 × 10⁶ M⁻¹ s⁻¹ at pH 7.4 and 25 °C, respectively) and peroxynitrite (k = 1.0 × 10⁶ and k = 1.8 × 10⁷ M⁻¹ s⁻¹ at pH 7.4 and 25 °C, respectively). The reductive part of the catalytic cycle was also studied, and the rate constant for the reduction of c-TXNPx by tryparedoxin I was 1.3 × 10⁶ M⁻¹ s⁻¹. The catalytic role of two conserved cysteine residues in both TXNPxs was confirmed with the identification of Cys52 and Cys173 (in c-TXNPX) and Cys81 and Cys204 (in m-TXNPx) as the peroxidatic and resolving cysteines, respectively. Our results indicate that mitochondrial and cytosolic TXNPxs from T. cruzi are highly efficient peroxidases that reduce hydrogen peroxide and peroxynitrite, and contribute to the understanding of their role as virulence factors reported in vivo.     

Involvement of malate,monophenols, and the superoxide radical in hydrogen peroxide formation by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Peroxidase associated with isolated horseradish cell walls catalyzes the formation of H₂O₂ in the presence of NADH. The reaction is stimulated by various monophenols, especially of coniferyl alcohol. NADH can be provided by a bound malate dehydrogenase. This system is capable of polymerizing coniferyl alcohol yielding an insoluble dehydrogenation polymer. NADH was found to be oxidized by two different mechanisms, one involving Mn²⁺, monophenol, and the superoxide radical O₂ ^·- in a reaction that is not affected by superoxide dismutase, and another one depending on the presence of free O₂ ^·- and probably of an enzyme-NADH complex. A scheme of these reaction chains, which are thought to be involved in the lignification process, is presented.Abbreviations DHP dehydrogenation polymer - GOT glutamate oxaloacetate transaminase (EC 2.6.1.1) - LDH lactate dehydrogenase (pig heart, EC 1.1.1.27) - MDH malate dehydrogenase (EC 1.1.1.37) - pCA p-coumaric acid - SOD superoxide dismutase (EC 1.15.1.1) - TLC thin-layer chromatography - XOD xanthine oxidase (EC 1.2.3.2)