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1.
A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.  相似文献   

2.
Three turnip peroxidases (fractions C1, C2, and C3) were partially purified and characterized, to permit study of their feasibility for use in clinical and enzyme immunoassays. These fractions represented 20% of the initial activity, and fractions C1 and C2 were purified to homogeneity. The optimum pH was between 5.0 and 5.5, while optimum temperature ranged from 40 to 55 degrees C. The ABTS K(m) values for the two acidic fractions (C2 and C3) were 0.70 and 0.42 mM, respectively; about 5 times lower than that reported for the acidic commercial horseradish peroxidase (HRP). Fraction C3 had 4 times higher K(m) value than commercial cationic HRP. The molecular weights determined by SDS-PAGE ranged from 39.2 to 42.5 kDa. Activation energies for inactivation were 113 (C1), 130 (C2), and 172 kJ/mol (C3) which are higher or comparable to other peroxidase isoenzymes reported. Fractions C1 and C3 represent an alternative source of peroxidase because of their higher purification yield and specific activity, when compared to fraction C2.  相似文献   

3.
Structural changes involved in the reactivation of peroxidases (PODs) from broccoli and horseradish (HRP) following heat denaturation were investigated by using circular dichroism and absorption spectroscopy. Cooling heat-treated enzymes resulted in rapid refolding of the secondary structure into an inactive structural species, similar in conformation to the native enzyme. Reassociation of heme to the refolded peroxidase, as well as molecular rearrangement of the structure around the heme, occurs during incubation at approximately 25 degrees C and results in the return of biological activity. The secondary structure of neutral broccoli POD (N) is relatively heat labile, resulting in a rapid loss of activity, but the level of reactivation is high because the structure at the heme pocket is relatively stable. Acidic broccoli POD and HRP are more heat stable than N, but have a low degree of reactivation. Loss of activity is due primarily to alteration of the structure at the heme pocket. Effects of bovine serum albumin and pH on reactivation of PODs are also discussed. Keywords: Peroxidase; reactivation; horseradish; broccoli; circular dichroism; absorption spectroscopy.  相似文献   

4.
The partial characterization of an anionic peroxidase in melon fruit is described. Four melon peroxidase (MPX) isoenzymes were detected in crude extracts after isoelectric focusing. The major MPX isoenzyme (pI = 3.7) was partially purified by including hydrophobic and anion-exchange chromatography in the purification scheme. The sample obtained was used to characterize MPX. This peroxidase did not show activity on ascorbic acid but oxidized guaiacol at a high rate, showing an optimum pH of 5.5 when acting on this last reducing substrate. Melon fruits grown under highly saline conditions showed slightly increased levels of this anionic isoenzyme. Kinetic studies using 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) as reducing substrate showed that increased salinity in the growth medium did not modify the kinetic parameters of melon peroxidase on both hydrogen peroxide and reducing substrate.  相似文献   

5.
Turnip (Brassica napus) roots peroxidase isoforms have been used in diagnostic kits and can also efficiently polymerize phenolic compounds from wastewaters. Heterologous expression of a turnip acidic peroxidase (BnPA) was investigated to increase availability of this widely used enzyme. The mature BnPA was ligated into the pET28a(+) vector and used to transform Escherichia coli Rosetta 2. Recombinant BnPA peroxidase was overexpressed and accumulated in inclusion bodies from which it was purified to homogeneity by immobilized metal affinity chromatography under denaturing conditions. Peroxidase activity was observed after a refolding process under oxidative conditions. The yield of pure recombinant BnPA was 29 mg L(-1) of culture with a specific activity of 981 ± 20 ABTS units mg(-1) at optimal conditions (pH 6, 45 °C). Recombinant BnPA showed similar kinetic properties compared to native turnip peroxidase, and its secondary structure evaluated by circular dichroism comprised 20% α-helix, 32% β-sheet and 48% random structure. Recombinant BnPA showed high yield and good kinetic properties which are key steps for future structure-function studies and biotechnological applications.  相似文献   

6.
An acidic peroxidase (pI approximately 2.5) was purified from turnip roots (TAP), and its thermal properties were evaluated. TAP is a monomeric protein having a molecular weight (MW) of 49 kDa and a carbohydrate content accounting for 18% of the MW. The yield of pure TAP was relatively high ( approximately 2 mg/kg of fresh roots), with a specific activity of 1810 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) units/mg at pH 6. The activity increased 4-fold at the optimum pH (4.0) to 7250 ABTS units/mg, higher than that of most peroxidases. TAP was heat stable; heat treatment of 25 min at 60 degrees C resulted in 90% initial activity retention, whereas an activity of 20% was retained after 25 min of heating at 80 degrees C. TAP regained 85% of its original activity within 90 min of incubation at 25 degrees C, following heat treatment at 70 degrees C for 25 min. Thermal inactivation caused noticeable changes in the heme environment as evaluated by circular dichroism and visible spectrophotometry. TAP was rapidly denatured by heating in the presence of 1.0 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but the Soret band and activity were fully recovered by adding an excess of Ca(2+). This is further evidence that Ca(2+) plays an important role in the stability of TAP. The high specific activity of TAP, together with its relatively high thermal stability, has high potential for applications in which a thermally stable enzyme is required.  相似文献   

7.
The effect of 6-benzylaminopurine (6-BA) on the color, antioxidant activity, and contents of total phenols, glucosinolate, and sulforaphane in broccoli florets was investigated. The results showed that 6-BA treatment markedly inhibited the increase of the L* value and malondialdehyde (MDA) content and retarded the decrease of the H value. 6-BA treatment reduced the rate of chlorophyll degradation by regulating the activities of chlorophyllase and Mg-dechelatase. When compared to control florets, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) were enhanced in florets treated with 6-BA, whereas the activity of peroxidase (POD) was significantly reduced. The contents of total phenols, glucosinolate, and sulforaphane in broccoli florets were also profoundly increased after treatment with 6-BA. These results indicated that 6-BA could maintain the quality, delay senescence, and improve the nutritional value of broccoli.  相似文献   

8.
Cystine lyase is the enzyme responsible for off-aroma deterioration in fresh unblanched broccoli. In this research, cystine lyase purification from broccoli has been optimized. Only one protein peak with cystine lyase activity was found during purification. Broccoli cystine lyase was purified 100-fold to homogeneity. L-Cystine, L-cysteine-S-sulfate, L-djenkolic acid, and some S-alkyl-L-cysteines and their sulfoxides are substrates, but the enzyme had negligible activity with L-cystathionine. A K(m) value of 81.2 microM was found for L-cystine. Inhibition and K(i) determinations indicated that L-cysteine is a linear noncompetitive inhibitor with a K(i) of 5 mM and DL-homocysteine is a competitive inhibitor with a K(i) of 1.5 mM. The molecular weight of cystine lyase was determined to be 100 kDa by three methods, with two subunits of 48 kDa each and a carbohydrate content of 3%. Further characterization included cofactor quantification, the effects of temperature and pH on activity and stability, and amino acid composition.  相似文献   

9.
Using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate, it has been shown that the increased peroxidase activity for decreasing pH of myoglobin activated by hydrogen peroxide is due to a protonization of ferrylmyoglobin, MbFe(IV)=O, facilitating electron transfer from the substrate and corresponding to pK(a) approximately 5.2 at 25.0 degrees C and ionic strength 0.16, rather than due to specific acid catalysis. On the basis of stopped flow absorption spectroscopy with detection of the radical cation ABTS(.+), the second-order rate constant and activation parameters for the reaction between MbFe(IV)=O and ABTS were found to have the values k = 698 +/- 32 M(-1) s(-1), DeltaH# = 66 +/- 4 kJ mol(-1), and DeltaS# = 30 +/- 15 J mol(-1) K(-1) at 25.0 degrees C and physiological pH (7.4) and ionic strength (= 0.16 M NaCl). At a lower pH (5.8) corresponding to the conditions in meat, values were found as follows: k = 3.5 +/- 0.3 x 10(4) M(-1) s(-1), DeltaH# = 31 +/- 6 kJ mol(-1), and DeltaS# = -53 +/- 19 J mol(-1) K(-1), indicative of a shift from outersphere electron transfer to an innersphere mechanism. For steady state assay conditions, this shift is paralleled by a shift from saturation kinetics at pH 7.4 to first-order kinetics for H2O2 as substrate at pH 5.8. In contrast, the activation reaction between myoglobin and hydrogen peroxide was found at 25.0 degrees C to be slow and independent of pH with values of 171 +/- 7 and 196 +/- 19 M(-1) s(-1) found at physiological and meat pH, respectively, as determined by sequential stopped flow spectroscopy, from which a lower limit of k = 6 x 10(5) M(-1) s(-1) for the reaction between perferrylmyoglobin, .MbFe(IV)=O, and ABTS could be estimated. As compared to the traditional peroxidase assay, a better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.  相似文献   

10.
《Soil biology & biochemistry》2001,33(7-8):1021-1028
The adsorption, desorption, catalytic activity, and susceptibility to microbial degradation of the enzyme horseradish peroxidase (HRP E.C. 1.11.1.7), on Wyoming montmorillonite (M) homoionic to Na+ or Ca2+ were investigated. Adsorption at equilibrium was reached after 1 h of contact between the clay and HRP. The adsorption isotherms were of the L type and fitted the Freundlich equation on M-Na and the Langmuir equation on M-Ca. Adsorption was greater on M-Na than on M-Ca and was maximal at pH 3.0, i.e., below the isoelectric point (pI=pH 9) of the protein. Only 10–25% of HRP was desorbed from the equilibrium M-Na-HRP complexes, whereas 20–30% was desorbed from the equilibrium M-Ca-HRP complexes with 4–7 washes with double distilled water. HRP partially penetrated the interlayers of M-Na and M-Ca, but complete intercalation was observed only at pH 3. The enzymatic activity of HRP measured immediately after the preparation of the complexes at all concentrations was greatly reduced when bound on M-Na (about 90%), regardless of the loading of HRP. The reduction in activity of M-Ca-HRP was related to the amount of bound protein (no reduction for the highest and 60% for the lowest amount bound). After 24 h, pure HRP in dilute solution lost about 10% of its catalytic activity daily, whereas when bound on M-Ca, a greater reduction was observed (about 30% for the highest and 60% for the lowest amount bound). FT-IR analyses indicated only small changes in the secondary structure of HRP as a result of binding on the clays. Electronic absorption spectra in the UV region of bound HRP did not show the typical ‘red-shift’ of the Soret band that usually results from binding of HRP with its substrate. Consequently, the reduction in the activity of bound HRP was probably the result of the inaccessibility and/or of modifications of the active center of HRP for its substrate. The availability of HRP bound on M-Na as a source of carbon and/or nitrogen for soil microorganisms was reduced by 90% in comparison with the free enzyme.  相似文献   

11.
Heat inactivation characteristics differed for acidic (A), neutral (N), and basic (B) broccoli peroxidase. At 65 degrees C, A was the most heat stable followed by N and B. The activation energies for denaturation were 388, 189, and 269 kJ/mol for A, N, and B, respectively. Reactivation of N occurred rapidly, within 10 min after the heated enzyme was cooled and incubated at room temperature. The extent of reactivation varied from 0 to 50% depending on the isoenzyme and heating conditions (temperature and time). The denaturation temperature allowing the maximum reactivation was 90 degrees C for A and horseradish peroxidase (HRP) and 70 and 80 degrees C for B and N, respectively. In all cases, heat treatment at low temperatures for long times prevented reactivation of the heated enzymes. Calcium (5 mM) increased the thermal stability of N and B but had no effect on reactivation. The presence of 0.05% bovine serum albumin decreased thermal stability but increased the extent of reactivation of A..  相似文献   

12.
The extract of soybean exposed to biotic elicitors such as food-grade fungus is known to have antioxidant activity. Glyceollins were major bioactive compounds present in soybean elicited by fungi and shown to have antifungal and anticancer activities. The purpose of present study was to evaluate the antioxidant activities of glyceollins by measuring ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, singlet oxygen quenching, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, hydroxyl radical scavenging activity, and lipid peroxidation inhibition. In addition, the antioxidant potential of glyceollins were measured by a fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFDA), and dihydroethidium (DHE) in mouse hepatoma hepa1c1c7 cells in which they were insulted with H2O2 to generate reactive oxygen species (ROS). Glyceollins showed a strong reducing power and inhibited lipid peroxidation, with significant scavenging activities of radicals including singlet oxygen, superoxide anion, ABTS, and DPPH. We also found that glyceollins significantly suppressed H2O2-induced ROS production in hepa1c1c7 cells. Therefore, glyceollins deserve further study as natural antioxidants and nutraceuticals.  相似文献   

13.
A new analytical method (liquid chromatography-antioxidant, LC-AOx) was used that is intended to separate beer polyphenols and to determine the potential antioxidant activity of these constituents after they were allowed to react online with a buffered solution of the radical cation 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(?+)). Using the LC-AOx method, it was possible to demonstrate that the extent of the antioxidant activity was very much dependent on the phenolic compound considered. The method was also applied to the analysis of beer extracts and allowed the evaluation of their antioxidant activity at different steps of beer processing: brewing, boiling, and fermentation. This study showed that the total antioxidant activity remained unchanged throughout beer processing, as opposed to the polyphenolic content, which showed a 3-fold increase. Hopping and fermentation steps were the main causes of this increase. However, the increase measured after fermentation was attributed to a better extraction of polyphenols due to the presence of ethanol, rather than to a real increase in their content. Moreover, this method allowed the detection of three unknown antioxidant compounds, which accounted for 64 ± 4% of the total antioxidant activity of beer and were individually more efficient than caffeic acid and epicatechin.  相似文献   

14.
为探讨6-苄氨基嘌呤（6-Benzylaminopurine,6-BA）处理对鲜切西兰花中硫代葡萄糖苷（简称硫苷）代谢的作用机理,该研究首先鉴定得出西兰花花球中分别以萝卜硫苷和萝卜硫素为主的12种硫苷和6种水解产物,然后以30 mg/L的6-BA溶液对鲜切西兰花进行处理,研究外源6-BA处理对其硫苷含量、黑芥子酶活性、硫苷水解产物及其代谢途径关键基因表达水平等的影响。结果表明,6-BA处理通过延缓西兰花花球叶绿素含量下降从而延迟其黄化进程;同时,显著提高了脂肪族硫苷合成相关基因MYB28、CYP79F1、CYP83A1、ST5b、FMOGS-OX1,和吲哚族硫苷合成关键基因MYB51、CYP79B2及CYP83B1的表达水平（P<0.05）,其中6-BA处理组FMOGS-OX1的表达量在贮藏第6天时为对照组的1.93倍,MYB51的表达量为对照组1.41～1.91倍,CYP79B2的表达量在4 d后高出对照组41.90%～93.75%;由此,6-BA处理保持了其组织较高的萝卜硫苷、3-甲基亚磺酰丙基硫苷、吲哚甲基硫苷和4-甲氧基吲哚甲基硫苷的含量,总硫苷含量为对照组的2.46～4.79倍;此外,6-BA处理的西兰花花球组织黑芥子酶活性和其基因MYR的表达水平在4～8 d期间分别高出对照组15.32%～90.22%和25.86%～33.73%,并显著提高了其组织萝卜硫素等异硫氰酸酯水解产物的含量（P<0.05）,其中萝卜硫素含量高于对照组17.58%～25.39%,但对硫苷水解关键基因ESP的表达量并无显著影响（P>0.05）。因此,6-BA处理可通过提高硫苷合成相关基因和水解关键基因来保持鲜切西兰花花球中硫苷的含量和异硫氰酸酯的水平,为西兰花功能活性成分的保持提供理论和技术支持。  相似文献   

15.
Pepsin proteolysis at pH approximately 4 resulted in a lowering of the (pseudo)peroxidase activity of metmyoglobin both at physiological pH and at meat pH, as measured by a peroxidase assay with H(2)O(2) and ABTS as substrates. In contrast, the mildly proteolyzed myoglobin had a strongly enhanced prooxidative effect on lipid oxidation in an oil in water methyl linoleate emulsion compared to native metmyoglobin, as evidenced by rates of oxygen depletion. More severe proteolysis of metmyoglobin at lower pH values near the optimum for pepsin did not result in a similar enhancement of prooxidative activity. The mildly proteolyzed metmyoglobin had spectral characteristics in agreement with a relative stabilization of the iron(II) state. On the basis of the observed effects of metal chelators, of lipophilic and hydrophilic peroxides and of radical scavengers on oxygen depletion rates, it is suggested that the increased prooxidative effect is due to radicals formed by cleavage of lipid peroxides by iron(II)/iron(III) cycling of a heme pigment with affinity for the lipid/water interface.  相似文献   

16.
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17.
Both selenium and allophycocyanin (APC) have been reported to show novel antioxidant activities. In this study, a fast protein liquid chromatographic method for purification of selenium-containing allophycocyanin (Se-APC) from selenium-enriched Spirulina platensis and the protective effect of Se-APC on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress have been described. After fractionation by ammonium sulfate precipitation, and separation by DEAE-Sepharose ion-exchange and Sephacryl S-300 size exclusion chromatography, Se-APC with purity ratio (A652/A280) of 5.30 and Se concentration of 343.02 μg g(-1) protein was obtained. Se-APC exhibited stronger antioxidant activity than APC by scavenging ABTS (2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid) and AAPH free radicals. The oxidative hemolysis and morphological changes induced by AAPH in human erythrocytes were effectively reversed by coincubation with Se-APC. Lipid oxidation induced by the pro-oxidant agent cupric chloride in human plasma, as evaluated by formation of conjugated diene, was blocked by Se-APC. The accumulation of malondialdehyde, loss of reduced glutathione, and increase in enzyme activities of glutathione peroxidase and reductase induced by AAPH in human erythrocytes were effectively suppressed by Se-APC. Furthermore, Se-APC significantly prevented AAPH-induced intracellular reactive oxygen species (ROS) generation. Taken together, our results suggest that Se-APC demonstrates application potential in treatment of diseases in which excess production of ROS acts as a casual or contributory factor.  相似文献   

18.
为高效利用美国豆芋花皂苷类化合物,以皂苷得率和纯度为指标,研究美国豆芋花总皂苷的提取工艺及大孔树脂纯化工艺,并对制备的美国豆芋花总皂苷的抗氧化及α-葡萄糖苷酶活性抑制能力进行研究。结果表明,美国豆芋花总皂苷的提取工艺为提取溶剂90%乙醇,料液比1∶20,温度70℃,提取时间2 h。得到的提取物石油醚脱脂后用水饱和正丁醇萃取,D-101大孔树脂吸附柱纯化的工艺为上样皂苷质量浓度1 mg·m L~(-1),上样量40 m L·g~(-1)树脂,洗脱剂为80%乙醇(pH值6),洗脱剂用量4 BV(树脂柱体积)。制备的美国豆芋花总皂苷样得率4.24%,纯度达65.35%,比粗提物(纯度15.27%)提高了约3倍;抗氧化性能(清除DPPH和ABTS自由基IC50值分别为51.08、29.24μg·m L~(-1))和α-葡萄糖苷酶活性抑制能力(IC50值为83.62μg·m L~(-1))也均优于粗提物(清除DPPH、ABTS自由基IC50值分别为286.51、69.92μg·m L~(-1),α-葡萄糖苷酶的抑制浓度(IC50)值为1 043.57μg·m L~(-1)),表明该工艺可用于高活性美国豆芋花总皂苷的富集和纯化。此外,美国豆芋花总皂苷抑制α-葡萄糖苷酶活性的抑制类型为竞争型抑制,抑制常数为49.68μg·m L~(-1)。本研究结果为开发具有降血糖功能的医药中间体或功能性食品提供了新资源和实践基础。  相似文献   

19.
Tannin-protein complexes as radical scavengers and radical sinks   总被引：8，自引：0，他引：8  
The 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonic acid) radical cation (ABTS(*)(+)) decolorization assay has been used to determine the antioxidant activity of the polyphenol epicatechin(16) (4 --> 8) catechin (procyanidin, PC) alone or in complex with the model proteins bovine serum albumin (BSA) or gelatin. PC had a molar antioxidant capacity of approximately 54, 92, or 108 radicals at pH values of 3.0, 4.9, or 7.4, respectively. Radical scavenging occurred via a rapid step followed by a slow step. Interaction with gelatin reduced the rate of rapid scavenging by 50% (PC-BSA mixtures reduced by 15%). Inhibition paralleled formation of precipitable PC-protein complexes over a range of protein/PC ratios. However, inhibition was virtually overcome in 10 min. Reaction with ABTS(*)(+) converted the PC-protein complexes from a dissociable form to a form resistant to dissociation by strong denaturants such as SDS. This study demonstrates that PC is a potent ABTS(*)(+) scavenger even when bound to protein and that the complexes may act as a radical sink within the gastrointestinal tract.  相似文献   

20.
The objective of the study was to assess the antioxidant potential of alcalase-treated zein hydrolysate (ZH) during a two-stage (1 h of pepsin --> 0.5-2 h of pancreatin, 37 degrees C) in vitro digestion. Sephadex gel filtration and high-performance size exclusion chromatography were used to separate ZH into fractions. The amino acid composition, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+*)) and 1,1-diphenyl-2-picrylhydrazyl (DPPH*) free radical scavenging activity, reducing power, and Cu (2+) chelation ability were tested to determine the antioxidant efficacy of ZH. Results showed that in vitro digests of ZH contained up to 16.5% free amino acids, with short peptides (<500 Da) making up the rest of the mass. The ABTS(+*) scavenging activity of ZH was decreased by 27% (P<0.05) after pepsin treatment but was fully recovered upon subsequent pancreatin digestion, while the DPPH* scavenging activity of ZH was substantially less than ABTS(+*) scavenging activity and showed a 7-fold reduction following pancreatin treatment. The reducing power of ZH increased 2-fold (P<0.05) following pancreatin digestion when compared with nondigested ZH. The ability of ZH to sequester Cu (2+) was reduced by pepsin digestion but was reestablished following pancreatin treatment. The antioxidant activity demonstrated by in vitro digests of ZH (1-8 mg/mL) was comparable to or exceeded (P<0.05) that of 0.1 mg/mL of ascorbic acid or BHA. The results suggested that dietary zein alcalase hydrolysate may have the benefit to promote the health of the human digestive tract.  相似文献   

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