Effect of Additives on the Inactivation of Lysozyme Mediated by Free Radicals Produced in the Thermolysis of 2, 2-Azo-Bis-(2-Amidinopropane)

The inactivation of lysozyme caused by the radicals produced by thermolysis of 2, 2-azo-bis-2-amidino-propane can be prevented by the addition of different compounds that can react with the damaging free radicals. Compounds of high reactivity (propyl gallate, Trolox, cysteine, albumin, ascorbate, and NADH) afford almost total protection until their consumption, resulting in well-defined induction times. The number of radicals trapped by each additive molecule consumed ranges from 3 (propyl gallate) to 0.12 (cysteine). This last value is indicative of chain oxidation of the inhibitor. Uric acid is able to trap nearly 2.2 radicals per added molecule, but even at large (200 μM) concentrations, a residual inactivation of the enzyme is observed, which may be caused by urate-derived radicals.

Compounds of lower reactivity (tryptophan, Tempol, hydroquinone, desferrioxamine, diethylhydroxylamine, methionine, histidine, NAD⁺ and tyrosine) only partially decrease the lysozyme inactivation rates. For these compounds, we calculated the concentration necessary to reduce the enzyme inactivation rate to one half of that observed in the absence of additives. These concentrations range from 9 μM (tryptophan and Tempol) to 5 mM (NAD⁺).     

Effects of Oxygen Radical Scavengers on the Inactivation of SS ΦX174 DNA by the Semi-Quinone Free Radical of the Antitumor Agent Etoposide

We have studied the effects of oxygen radical scavengers on the inactivation of ss ΦX174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH ≥ 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss ΦDX174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO. the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH ≥ 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH· trapping but from trapping of O₂^-. DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH ≥ 7.4. Potassium superoxide also stimulated ΦDX174 DNA inactivation by the ortho-quinone at pH ≤ 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the orthoquinone of etoposide, thus indirectly influencing DNA inactivation.     

Esr and Spin-Trapping Study of Free Radicals in γ-Irradiated Solid Lysozyme

Free radicals produced by γ-irradiation of solid lysozyme were investigated by a technique combining ESR, spin-trapping and enzymatic digestion. MNP and DMPO were used as spin-trapping reagents. The solid lysozyme was first γ-irradiated and then dissolved in an aqueous solution containing the spin-trapping reagent to stabilize free radicals. The spin adducts of lysozyme were digested to oligopeptides to get ESR spectra having a well-resolved hyperfine structure. The ESR spectra obtained showed that carbon-centered radicals, CH-, at the side chains of amino acids, and thiyl radicals, -CH₂-_-S^-, at disulfide bridges were produced in γ-irradiated solid lysozyme.     

Role of Oxygen Free Radicals in Respiratory Distress Induced by Arachidonic Acid In the Rat

The purpose of our present study is the possible implication of oxygen free radicals in the respiratory distress induced in rats by intravenous administration of arachidonic acid (20mg/kg). The respiratory frequency was measured and plasma TXB2 concentration was assayed by RIA from blood withdrawn I min after arachidonic acid administration. The substances studied were: SOD, catalase, manifold, DMSO, BHT, imidazole. All the drugs, except imidazole, significantly protect the rats from the respiratory distress induced by arachidonic acid. SOD, catalase, BHT and imidatole inhibit whereas mannitol and DMSO increase the plasma levels of TXB2. We suggest that oxygen free radicals generated in the respiratory burst induced by arachidonic acid are mainly responsible for the consequent respiratory distress.     

超氧化物歧化酶在氧自由基所引起的癌变中的作用

超氧化物歧化酶是一类抗氧化酶,它能催化超氧阴离子自由基的歧化反应,对机体起保护作用。氧自由基在某些情况下会对机体产生损伤作用。文章就氧自由基的产生、氧自由基与致癌的关系,以及超氧化物歧化酶在氧自由基所引起的癌变中的作用等方面进行了综述。     

黄芪有效成分对氧自由基清除作用的ESR研究

用电子自旋共振技术研究了黄芪总黄酮(TFA)、黄芪总皂甙(TSA)和黄芪总多糖(TPA)对次黄嘌呤／黄嘌呤氧化酶体系产生的超氧阴离子自由基和H₂O₂－Fe²⁺体系产生的羟自由基的清除作用．结果表明,这3种成分均有清除氧自由基的作用;对超氧阴离子自由基的清除效能大于对羟自由基的清除作用;其作用强度依次为TFA＞TSA＞TPA．结果提示清除氧自由基可能是黄芪抗衰老的主要机理之一,TFA和TSA是黄芪抗氧化作用的主要药理活性成分．     

芥子碱硫氰酸盐清除超氧阴离子自由基作用的研究

目的:研究芥子碱硫氰酸盐清除超氧阴离子自由基的作用。方法:用化学发光法测定芥子碱硫氰酸盐清除超氧阴离子自由基的能力,并以抗坏血酸的清除能力做为对照,以IC50值(清除率为50%时的浓度值)作为评价指标。结果:芥子碱硫氰酸盐和抗坏血酸的IC50值分别为0.135 mmol/L和18.74 mmol/L,后者的IC50值约为前者的140倍。结论:芥子碱硫氰酸盐具有良好的抗氧化作用,可以作为天然抗氧化剂进行开发。     

过氧化心肌线粒体产生的脂类自由基及（－）－EGCG的抑制作用

本文利用ＥＳＲ技术研究了心肌线粒体酶修饰下的脂质过氧化和脂类自由基,以及（－）－ＥＧＣＧ的抑制作用。结果表明：４－ＰＯＢＮ能捕集ｌｉｐｏｘｙｇｅｎｇｓｅ诱发心肌线粒体产生的自由基,得到６条线谱的脂类自由基和４条线谱捕集物,（－）－ＥＧＣＧ对该体系中使用的１ｉｐｏｘｙｅｎａｓｅ活性无影响,对所产生的自由基有明显的清除作用,并呈量效关系。对脂质过氧化的抑制作用,在本试验浓度范围内随浓度增加变化不大,最大抑制率约２０％。     

不同南瓜多糖体外清除羟基自由基作用的研究

采用热水浸提法和超声波辅助法提取南瓜粗多糖,用十六烷基三甲基溴化铵(CTAB)络合沉淀得AP1多糖;用邻二氮菲-金属铁离子-H2O2体系检测南瓜多糖对羟基自由基的清除作用。结果表明,南瓜多糖能有效清除羟基自由基,并随着浓度的增加清除作用加强,且热水提取的南瓜多糖对羟基自由基清除作用显著高于超声提取的南瓜多糖。该结果表明南瓜多糖具有抗氧化性,并且热水提取的南瓜多糖的清除羟基自由基最为显著。     

Effect of Ascorbic Acid Supplementation on Certain Oxidative Stress Parameters in the Post Reperfusion Patients of Myocardial Infarction

Reperfusion injury causes oxidative stress thereby resulting in an imbalance between oxidant-antioxidant systems. In the present communication, the effect of ascorbic acid supplementation has been studied on certain oxidant and antioxidant parameters in the blood of the patients with myocardial infarction before and after thrombolysis. In patients after thrombolysis, the activity of antioxidant enzyme, superoxide dismutase, in the blood was found to be significantly reduced where as the activity of the oxidant enzyme, xanthine oxidase, was found to be significantly increased. Malondialdehyde levels, the index of free radical mediated damage, was also found to be significantly elevated in thrombolyzed patients compared to the patients before thrombolysis. Supplementation of vitamin C to the post reperfusion patients restored these parameters back to normal or near normal levels.     

Pharmacokinetic Analysis of Free Radicals by in vivo BCM (Blood Circulation Monitoring)-ESR Method

In pharmacokinetic studies, a variety of analytical method including radioisotopic detection and HPLC (high performance liquid chromatography) has been used. In the present investigation, we developed in vivo BCM (Blood Circulation Monitoring)-ESR method, which is a new technique with a conventional X-band ESR spectrometer for observing stable free radicals in the circulating blood of living rats under anaesthesia. Both 5-(PROXYL derivatives) and 6-(TEMPO derivatives) membered nitroxide spin probes with various types of substituent functional group were used. After physicochemical properties of the spin probes such as hyperfine coupling constant (A-value), g-value and partition coefficient as well as chemical stability of the compounds in the fresh blood were obtained, the in vivo BCM-ESR method was performed in normal rats. Several pharmacokinetic parameters such as half-life of the probes, distribution volume, total body clearance and mean residence time were obtained and discussed in terms of their chemical structures. In addition, clearance of a spin probe was related to the urine concentration. The BCM-ESR method was found to be very useful to observe free radicals at the real time. By time-dependent ESR signal decay of spin probes, pharmacokinetic parameters were obtained.     

Pharmacokinetic Analysis of Free Radicals by in vivo BCM (Blood Circulation Monitoring)-ESR Method

In pharmacokinetic studies, a variety of analytical method including radioisotopic detection and HPLC (high performance liquid chromatography) has been used. In the present investigation, we developed in vivo BCM (Blood Circulation Monitoring)-ESR method, which is a new technique with a conventional X-band ESR spectrometer for observing stable free radicals in the circulating blood of living rats under anaesthesia. Both 5–(PROXYL derivatives) and 6–(TEMPO derivatives) membered nitroxide spin probes with various types of substituent functional group were used. After physicochemical properties of the spin probes such as hyperfine coupling constant (A-value), g-value and partition coefficient as well as chemical stability of the compounds in the fresh blood were obtained, the in vivo BCM-ESR method was performed in normal rats. Several pharmacokinetic parameters such as half-life of the probes, distribution volume, total body clearance and mean residence time were obtained and discussed in terms of their chemical structures. In addition, clearance of a spin probe was related to the urine concentration. The BCM-ESR method was found to be very useful to observe free radicals at the real time. By time-dependent ESR signal decay of spin probes, pharmacokinetic parameters were obtained.     

自由基与软骨基质胶原蛋白类型改变

本文利用ＳＤＳ－聚丙烯酰胺凝胶电泳方法,定量研究了体外培养的软骨细胞和软骨组织基质中Ⅱ型胶原蛋白的含量。结果表明氧自由基（·Ｏ－２和·ＯＨ）和具有自由基性质的物质（黄腐酸,镰刀菌毒素）可使软骨细胞合成,分泌异常的非Ⅱ型的胶原蛋白,同时,硒化合物可明显地抑制此种效应。     

钙过负荷和丹参酮对线粒体脂质过氧化的影响

用ＥＳＲ自旋捕集技术捕捉到Ｆｅ２＋启动的心肌线粒体膜脂质过氧化过程中产生的脂类自由基Ｌ·（ａＮ＝１５．６Ｇ,ａＨ＝２．９７Ｇ）。钙过负荷时,钙离子使线粒体产生的脂类自由基增加,当钙浓度为３０μｍｏｌ／Ｌ·ｍｇＰｒ时,产生的脂类自由基约增加３２％。丹参酮可清除Ｆｅ３＋启动的线粒体膜脂质过氧化过程中产生的脂类自由基。当丹参酮浓度为０．８ｍｇ／ｍｇＰｒ时,清除率达到７０％左右。钙过负荷时丹参酮仍能有效清除脂类自由基。另外,还发展了用自旋探针ＣＴＰＯ测量线粒体氧消耗的方法,检测氧消耗所需样品量比Ｃｌａｒｋ电极少几十倍。用此方法测量小鼠心肌线粒体不同状态的氧消耗和呼吸控制率,证明一定浓度的Ｃａ２＋和Ｆｅ２＋影响线粒体的呼吸功能．     

Effect of Short-Term Caloric Restriction on H2O2 Production and Oxidative DNA Damage in Rat Liver Mitochondria and Location of the Free Radical Source

Oxygen free radicals (ROS) of mitochondrial origin seem to be involved in aging. Whereas in other tissues complexes I or III of the respiratory chain contain the ROS generators, in this study we find that rat liver mitochondria generate oxygen radicals at complexes I, II, and III. Short-term (6 weeks) caloric restriction significantly decreased H₂O₂ production in rat liver mitochondria. This decrease in ROS production was located at complex I because it occurred with complex I-linked substrates (pyruvate/malate), but did not reach statistical significance with the complex II-linked substrate succinate. The mechanism responsible for the lowered ROS production was not a decrease in oxygen consumption. Instead, the mitochondria of caloric-restricted animals released less ROS per unit electron flow. This was due to a decrease in the degree of reduction of the complex I generator. Furthermore, oxidative damage to mitochondrial and nuclear DNA was also decreased in the liver by short-term caloric restriction. The results agree with the idea that caloric restriction delays aging, at least in part, by decreasing the rate of mitochondrial ROS generation and thus the rate of attack to molecules, like DNA, highly relevant for the accumulation of age-dependent changes.     

建宁莲子糖蛋白的分离、纯化及清除自由基作用

从红花建莲(Nelumbo nucifera Gacrtn.Fujian)的莲子中提取出具有生物活性的糖蛋白(GLP),并进行清除自由基作用的研究。用60％(NH^4)2SO4饱和溶液沉淀蛋白质,经ConA-Sepharose 4B亲和层析校分离,SephadexG-150凝胶层析校纯化,得到2个组分的糖蛋白,分子量分别为25kD的GLP-I和92．5kD的GLP—E,后者为其主要成分。GLP中蛋白质含量是58．8％,总糖含量是36．4％。TLC和GC分析结果：GLP—I是由L—鼠李糖,D—木糖,D—甘露糖和D—葡萄糖组成。其中性糖摩尔比：1．08：0．92：2．54：4．15。GLP—E是由L—鼠李糖D—木糖,D—甘露糖,L—阿拉伯糖,D—半乳糖,D—葡萄糖和D—葡萄糖醛酸组成,其中性糖摩尔比：1．25：1．03：2．85：0．94：3．1634．65。药理实验表明：两种糖蛋白均有清除自由基的作用,而GLP—Ⅱ清除能力大于GLP—I。     

Block of Brain Sodium Channels by Peptide Mimetics of the Isoleucine,Phenylalanine, and Methionine (IFM) Motif from the Inactivation Gate

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH₂, measured on sodium channels whose inactivation was slowed by the α-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH₂ with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH₂ did not cause appreciable fast block, but the divalent cation IFM-NH(CH₂)₂NH₂ was as effective as the pentapeptide ac-KIFMK-NH₂. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH₂ showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.     

Damage to cultured lens epithelial cells of squirrels and rabbits by UV-A (99.9%) plus UV-B (0.1%) radiation and alpha tocopherol protection

The purpose of this research is to observe the near-UV radiation induced damage to cultured rabbit and squirrel lens epithelial cells as related to destruction and alterations of specific biochemical targets in the cells and to determine protective effects on the cells and targets that are provided by -tocopherol.Confluent monolayers of cultured rabbit and squirrel lens epithelial cells were exposed to black light (BL) lamps, which emit predominantly UV-A radiation. These cells received a mixture 3 J/cm² of UV-A and 4 mJ/cm² of UV-B per h. This mixture is termed near UV_A (ie:predominantly UV-A). Cells were exposed in Tyrode's or in MEM without or with -tocopherol added at 2.5–10 g/ml. Analyses of cell viability and survival, the physical state of cytoskeletal actin, and the activities of Na-K-ATPase and catalase were made.Exposure to near UV_A damaged these cells as measured by vital staining and colony forming ability. Pretreatment with -tocopherol decreased the magnitude of near UV_A cytotoxicity. Near UV_A exposure in MEM always produced more damage to the cells and biochemical targets than in Tyrode's. Cytoskeletal actin was degraded and the activities of Na-K-ATPase and catalase were markedly inhibited by UV-exposure. All of these targets were at least partially protected by -tocopherol in the medium. Without -tocopherol added to the media, the viability and survival of the cells did not recover even after 25 h of incubation. Cell viability was better protected from near UV_A by -tocopherol than was the ability to grow into colonies. This indicates that -tocopherol protects actin, catalase, and Na-K-ATPase from near UV_A damage.     

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: oxidative damage by hydroxyl free radicals and its prevention

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.