Showing papers in "Methods in Enzymology in 1984"
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TL;DR: In this article, the catalytic activity of catalase has been investigated using ultraviolet (UV) spectrophotometry and Titrimetric methods, which is suitable for comparative studies for large series of measurements.
Abstract: Publisher Summary Catalase exerts a dual function: (1) decomposition of H 2 O 2 to give H 2 O and O 2 (catalytic activity) and (2) oxidation of H donors, for example, methanol, ethanol, formic acid, phenols, with the consumption of 1 mol of peroxide (peroxide activity) The kinetics of catalase does not obey the normal pattern Measurements of enzyme activity at substrate saturation or determination of the K s is therefore impossible In contrast to reactions proceeding at substrate saturation, the enzymic decomposition of H 2 O 2 is a first-order reaction, the rate of which is always proportional to the peroxide concentration present Consequently, to avoid a rapid decrease in the initial rate of the reaction, the assay must be carried out with relatively low concentrations of H 2 O 2 (about 001 M) This chapter discusses the catalytic activity of catalase The method of choice for biological material, however, is ultraviolet (UV) spectrophotometry Titrimetric methods are suitable for comparative studies For large series of measurements, there are either simple screening tests, which give a quick indication of the approximative catalase activity, or automated methods
20,238 citations
TL;DR: Fixed-time assay measuring H 2 O 2 consumption and continuous monitoring of Glutathione disulfide (GSSG) formation are cited here.
Abstract: Publisher Summary To determine glutathione peroxidase reliably, some factors of potential pitfall have to be considered, for example, enzymatic side reactions of substrates (especially when crude tissue samples are assayed), high and variable spontaneous reaction rates of substrates, and the peculiar kinetics of the enzyme itself. With the best documented example, the enzyme of bovine red blood cells, ping-pong kinetics with infinite limiting maximum velocities, and Michaelis constants have been established. This means that the generally recommended conditions for determination of enzyme activity––that is, “saturating” concentrations of all substrates, cannot possibly be fulfilled. In consequence, compromises are inevitable in the choice of substrate concentration for the assay and in the definition of the unit of activity. Fixed-time assay measuring H 2 O 2 consumption and continuous monitoring of Glutathione disulfide (GSSG) formation are cited here. The main differences between the assay procedure described and those proposed by others are listed in the chapter. To compare the results obtained by different procedures, appropriate empirical converting factors are also given.
4,341 citations
TL;DR: This chapter presents a qualitative test for SOD activity based on the reduction of nitro blue tetrazolium (NBT) by O 2 · -, a simple immunological determination of the SOD molecule of sufficient sensitivity and avoiding labeled reagents, and an indirect measure of Sod activity to be used in purified samples.
Abstract: Publisher Summary The primary difficulty in assaying Superoxide dismutase (SOD) for its enzymatic activity consists in the free radical nature of its substrate O 2 · - which can only be supplied by generation within the assay medium. The substrate O 2 · - cannot easily be detected directly by conventional analytical tools. Routine testing of SOD, therefore, is performed according to a general principle, which is explained in the chapter. If an absolute measure of physiological levels of SOD is intended, a direct immunochemical method is suggested in addition to activity measurements. This chapter focuses on measuring SOD in biological media. It presents a qualitative test for SOD activity based on the reduction of nitro blue tetrazolium (NBT) by O 2 · - , a simple immunological determination of the SOD molecule of sufficient sensitivity and avoiding labeled reagents, and an indirect measure of SOD activity to be used in purified samples.
1,405 citations
TL;DR: The standard colorimetric and fluorometric methods are easily carried out using common laboratory equipment and are often adequate for routine analysis of vitamin E in animal tissues for biological research and for clinical testing.
Abstract: Publisher Summary A variety of methods have been developed by various investigators for the analysis of vitamin E in animal tissues, but many of the earlier procedures involving column chromotography and gas–liquid chromatography are rather complicated and time consuming. The most commonly used methods are based on the saponification and solvent extraction of lipids, the removal or destruction of interfering substances, and the determination of tocopherol spectrophotometricaily or spectrofluorometrically. The methods chosen are for common animal tissues, such as blood and organ tissues. A high-pressure liquid chromatography (HPLC) method for vitamin E has been introduced as a method of choice, but HPLC equipment is expensive and not readily available in every laboratory. The standard colorimetric and fluorometric methods are easily carried out using common laboratory equipment and are often adequate for routine analysis of vitamin E in animal tissues for biological research and for clinical testing. For more sophisticated and sensitive detection of commonly occurring α-tocopherol along with trace amounts of other forms of tocopherol and tocotrienol in animal tissues, the HPLC method is highly recommended.
879 citations
TL;DR: It was anticipated that platelet aggregation, if it occurs during the drawing of the blood, would liberate the TBA-reacting substances, and the effect of the aggregation was found to be eliminated by treatment with phosphotungstic acid–sulfuric acid system.
Abstract: Publisher Summary Because the amounts of lipid peroxides in the blood are rather small, a sensitive assay method is needed. For this purpose, the most appropriate among several reactions for detecting lipid peroxides is the thiobarbituric acid (TBA) reaction because of its sensitivity. TBA reaction with lipid peroxides gives a red-colored pigment. Malondialdehyde also gives the same product upon reaction with TBA. Because this product is fluorescent, a sensitive assay can be made by fluorometry. Results show that lipid peroxides can be measured by TBA reaction with fluorometry. Elimination of TBA-reacting substances other than lipid peroxides is necessary for the measurement of lipid peroxides in serum. The elimination procedure must be simple to avoid artifact due to the peroxidation during the procedure. One of the best procedures is to isolate lipids by precipitating them along with serum protein with the phosphotungstic acid–sulfuric acid system. By this procedure, water-soluble substances, which react with TBA to yield the same product as lipid peroxides, are removed. It was anticipated that platelet aggregation, if it occurs during the drawing of the blood, would liberate the TBA-reacting substances, and the effect of the aggregation was found to be eliminated by treatment with phosphotungstic acid–sulfuric acid system.
746 citations
TL;DR: Malonaldehyde is of interest primarily as a product of lipid peroxidation in vivo and as an index of oxidative rancidity in foods and reacts with a variety of compounds to form derivatives, which can be estimated spectrophotometricaily.
Abstract: Publisher Summary Malonaldehyde (MA) is of interest primarily as a product of lipid peroxidation in vivo and as an index of oxidative rancidity in foods. In biological materials, it exists in its free form and as a complex with various tissue constituents. It reacts with a variety of compounds to form derivatives, which can be estimated spectrophotometricaily. It has also been identified among the products of the oxidative decomposition of amino acids, complex carbohydrates, pentoses, and hexoses formed in the presence of a metal catalyst, as a product of free radicals generated by ionizing radiation in vivo, and as a byproduct of prostaglandin biosynthesis. However, peroxidation of fatty acids with three or more double bonds (notably arachidonic acid) is believed to be its major source. Because of its interest as an indicator of lipid peroxidation, various methods have been proposed for its estimation. The most widely employed method for the determination of MA in biological materials is based on its reaction with thiobarbituric acid (TBA).
540 citations
TL;DR: Mitochondrial O 2 - production is pH dependent and increases toward the alkaline region, and effective inhibitors of Mn-superoxide dismutase are not known, and matrical superoxide dismUTase is usually removed by the repetitive washing of submitochondrial particles obtained by sonication or by other means.
Abstract: Publisher Summary The determination of the rate of O 2 - production is based upon the spectrophotometric measurement of oxidation or reduction reactions in which O 2 - is a reactant. The concentration of the spectrophotometric indicator that reacts with O 2 - is adjusted to compete effectively with the spontaneous dismutation of O 2 - so that nearly all O 2 - produced can be detected. The involvement of O 2 - is ascertained by the use of superoxide dismutase that inhibits the reaction rate specifically due to O 2 - . Cyanide, which is often used as inhibitor of the mitochondrial cytochrome oxidase ( K i about 3 x 10 -5 M) is also an inhibitor of the often used copper-containing superoxide dismutase ( K i about 3 × 10 -4 M). It is possible to use enough cyanide that partially inhibits cytochrome oxidase without inhibiting superoxide dismutase. Alternatively, bacterial or mitochondrial (manganese-containing) superoxide dismutase can be used. Mitochondria have Mn-superoxide dismutase in the matrix space, so to measure the total production of O 2 - by mitochondrial membranes, the dismutase should be inhibited or removed. Because effective inhibitors of Mn-superoxide dismutase are not known, matrical superoxide dismutase is usually removed by the repetitive washing of submitochondrial particles obtained by sonication or by other means. Mitochondrial O 2 - production is pH dependent and increases toward the alkaline region.
527 citations
TL;DR: Ultraviolet spectrophometric detection of conjugated dienes has been used for many years in the food industry for the detection of autoxidized lipids and for a variety of pathological processes, the question has been raised whether peroxidative decomposition of membrane lipids has occurred in vivo.
Abstract: Publisher Summary Like many other substances, naturally occurring lipids exhibit simple end absorption in ultraviolet light as the wavelength is lowered toward 200 nm. The spectra of a variety of organic molecules containing conjugated dienes, however, are characterized by intense absorption, the so-called K band, which may range, with respect to peak absorption, from 215 to 250 nm, depending on nearby substituent groups. Ultraviolet spectrophometric detection of conjugated dienes has been used for many years in the food industry for the detection of autoxidized lipids. The method appears to have been applied for the first time to the problem of liver cell lipid peroxidation of toxigenic origin in 1966 and has been widely used since. For a variety of pathological processes, the question has been raised whether peroxidative decomposition of membrane lipids has occurred in vivo. A second principle of the method recognizes that for whole-animal studies involving possible lipid peroxidation, the fraction of endogenous lipids actually peroxidized may not only be low, but the process of lipid peroxidation may be confined to a particular subcellular structure.
454 citations
TL;DR: This chapter discusses the methods used for studying lipid peroxidation and concentrates on such aspects of lipidperoxidation in relation to biomembrane disturbance.
Abstract: Publisher Summary Lipid peroxidation is a complex process whereby unsaturated lipid material undergoes reaction with molecular oxygen to yield lipid hydroperoxides; in most situations involving biological samples, the lipid hydroperoxides are degraded to a variety of products, including aikanals, alkenals, hydroxyalkenals, ketones, and alkanes. Although attack by singlet oxygen on unsaturated lipid has been shown to give lipid hydroperoxide by a nonradical, nonchain process, the vast majority of situations involving lipid peroxidation proceed through a free radical-mediated chain reaction initiated by the abstraction of a hydrogen atom from the unsaturated lipid by a reactive free radical, followed by a complex sequence of propagative reactions. The peroxidation of polyunsaturated fatty acids (PUFAs) can proceed through nonenzymic autoxidative pathways or through processes that are enzymically catalyzed. The importance of autoxidation in the deterioration of foods, and in the oil industry, has long been recognized, and authoritative reviews are available for such aspects. A relatively new growth point for studies on lipid peroxidation has been the realization that many toxic agents can be metabolically activated within cells to free-radical intermediates that can initiate lipid peroxidation and result in cell injury. A very large number of such studies on lipid peroxidation in biological systems have demonstrated the degradation of membrane PUFAs, with a subsequent disorganization of membrane structure and disturbance of membrane function. This chapter discusses the methods used for studying lipid peroxidation concentrates on such aspects of lipid peroxidation in relation to biomembrane disturbance.
440 citations
TL;DR: This chapter describes the assay for the measurement of superoxide dismutase (SOD) activity which differs from the commonly used nitroblue tetrazolium (NBT)/cytochrome c SOD assays in the following ways.
Abstract: Publisher Summary This chapter describes the assay for the measurement of superoxide dismutase (SOD) activity which differs from the commonly used nitroblue tetrazolium (NBT)/cytochrome c SOD assays in the following ways. First of all, diethylenetriaminepentaacetic acid (DETAPAC) is used instead of ethylenediamine tetraacetic acid (EDTA). DETAPAC makes the SOD assay more sensitive, probably because Fe-DETAPAC does not react with O2·-, whereas Fe-EDTA does. Second, catalase is always included in present assay mixture, whereas most commonly used assays do not. Catalase is for two reasons: (1) H2O2 inhibits and even inactivates Cu,Zn-SOD, so the removal of H2O2 is necessary for preventing this inhibition and (2) for kinetic reasons, the assay should be run with the product (H2O2) as low as possible, so that the equilibrium will not be shifted in favor of O2·- production. In the assay procedure, varying concentrations of SOD activity are added until maximum inhibition is obtained. One unit of activity is that amount of protein that gives half-maximal inhibition. This assay differs from that of the cytochrome c method in that 95–100% inhibition is usually not achieved. With pure enzyme, 80–90% inhibition is obtained, whereas with most tissue homogenates, 70–80% inhibition is observed. This is why half-maximal inhibition is used rather than 50% inhibition as in the cytochrome c assay.
416 citations
TL;DR: In this article, the authors discuss the spin trapping of the biologically important free radicals: superoxide and hydroxyl, which can be detected using electron paramagnetic resonance (EPR).
Abstract: Publisher Summary Many free radicals of biological interest are highly reactive and never reach a concentration high enough to be detected by electron paramagnetic resonance (EPR). An example of this is the hydroxyl radical, which reacts with itself or with most organic molecules at diffusion-controlled rates. Its rate of reaction is limited mainly by the frequency with which it collides with other species. Thus, the direct detection of hydroxyl radicals by EPR in a biologic system is impossible. For short-lived radicals of lesser reactivity compared to the hydroxyl radical, there are various means of detection using EPR. A simple method is to slow the rate of disappearance of the radical by rapidly freezing the sample. This has the disadvantage that the radical is no longer in a fluid environment, and the resultant anisotropic effects can obscure the identification of the radical. In theory, spin trapping can overcome many of these difficulties. This technique consists of using a spin trap––that is, a compound that forms a stable free radical by reacting covalently with an unstable free radical. This chapter discusses the spin trapping of the biologically important free radicals: superoxide and hydroxyl.
TL;DR: O 2 - is the key intermediate in the generation of microbicidal oxidants by neutrophils through the action of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is a membrane-bound flavoprotein.
Abstract: Publisher Summary Neutrophils are phagocytic cells whose principal function is the destruction of invading bacteria. Bacterial killing is largely accomplished by means of an oxygen-dependent microbicidal system that employs, as antimicrobial agents, a group of lethal oxidants that the neutrophils generate when stimulated by bacterial targets. The precursor of these lethal oxidants is O 2 - . This substance is manufactured from oxygen by stimulated neutrophils and is subsequently converted into highly reactive lethal oxidants, such as OH· and HOCl, by a complicated series of secondary reactions. O 2 - is, therefore, the key intermediate in the generation of microbicidal oxidants by neutrophils. O 2 - is produced by neutrophils through the action of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. This oxidase, which is dormant in resting neutrophils but is activated when the cells are exposed to bacterial targets (or other suitable stimuli), is a membrane-bound flavoprotein. The oxidase can be assayed by measuring O 2 - production by neutrophil preparations. The measurement can be made using either intact neutrophils or broken cell preparations.
TL;DR: This chapter discusses the isolation, characterization, and detection of osteocalcin, a small protein comprising 10-20% of the noncollagenous protein in the bone.
Abstract: Publisher Summary This chapter discusses the isolation, characterization, and detection of osteocalcin Osteocalcin is a small protein (5800 daltons) comprising 10-20% of the noncollagenous protein in the bone Because osteocalcin is tightly adsorbed to the hydroxyapatite mineral phase of the bone, thorough extraction is best achieved by dissolving the mineral of finely pulverized bone Hydroxyapatite is soluble in neutral 05M ethylenediaminetetraacetic acid (EDTA), as well as in a variety of mineral and organic acids Uniformly high yields of 1-2 mg of osteocalcin per gram of dry bone are obtained with EDTA, whereas acid procedures often leave 10-25% of the protein behind, presumably because of precipitation during excursions through the osteocalcin isoelectric point The use of radioimmunoassay for the measurement of osteocalcin offers the advantages of specificity, sensitivity, and technical simplicity The method can easily detect nanogram quantities of the protein in bone extracts, cell cultures, and serum The assay is based on the competition of the radioactively labeled antigen and an identical nonlabeled antigen for binding to a specific antibody The amount of labeled antigen bound to the antibody is inversely proportional to the amount of unlabeled antigen present in the system
TL;DR: Production of O2- and H2O2 and their scavenging system are discussed in this chapter and are believed to be indispensable for the prevention of overreduction of electron carriers in the cyclic electron transport pathway.
Abstract: Publisher Summary In chloroplasts, chlorophyll-photosensitized production of 1O2 is an unavoidable reaction. Chloroplast thylakoids univalently photoreduce molecular oxygen producing O2- through the autoxidation of an electron acceptor in photosystem I. In chloroplasts, O2- produced in thylakoids is disproportionated by superoxide dismutases (SOD) in the stroma, and the H2O2 thus produced is reduced to water by ascorbate peroxidase. Ascorbate is regenerated from its oxidation products, dehydroascorbate (DHA) and monodehydroascorbate, by photoreductants through the system shown diagrammatically in this chapter. Ascorbate peroxidase, DHA reductase, and nicotinamide adenine dinucleotide (NADH)-dependent monodehydroascorbate reductase are localized in the chloroplast stroma, as are the enzymes participating in the generation of glutathione (GSH) and NADH. The production of O2- is enhanced under conditions where the generation rate of photoreductant in thylakoids exceeds that required for CO2 reduction; low CO2 concentration and high light intensity. O2- is, however, produced even when CO2 is supplied to chloroplasts and the photoproduction of O2- appears to be indispensable for the prevention of overreduction of electron carriers in the cyclic electron transport pathway. Thus, the photorcduction of O2- is an inevitable reaction in chloroplasts. Scavenging of O2- and H2O2 is essential for chloroplasts to maintain their ability to fix CO2, because several enzymes in the CO2-reduction cycle are sensitive to active oxygen. Production of O2- and H2O2 and their scavenging system are discussed in this chapter.
TL;DR: This chapter presents a sensitive, rapid method suitable for the extraction and determination of tocopherols in a range of small-tissue samples.
Abstract: Publisher Summary Free radical-catalyzed lipid peroxidation is a continual biological process, which if unchecked may cause damage to cellular and intracellular membranes, resulting not only in changed membrane structure but also in the destruction of the functional integrity of membrane-bound enzymes. Vitamin E, alone and in concert with the selenium-containing enzyme, glutathione peroxidase, can inhibit this process. This function of vitamin E may be aided by a specific physicochemical interaction between the phytyl side chain of α-tocopherol and the fatty acyl chains of polyunsaturated phospholipids, particularly those derived from arachidonic acid, by providing a mechanism for anchoring vitamin E to membranes and facilitating the antioxidant behavior attributed to α-tocopherol. Other consequences could be a reduction in permeability of biological membranes containing relatively high levels of polyunsaturated fatty acids, particularly arachidonic acid and the prevention of the degradation of membrane phospholipids by membrane-bound phospholipases in vivo . In recent years, a variety of methods have appeared in the literature for the high-pressure liquid chromatography (HPLC) determination of tocopherols in biological samples. Many of these have concerned themselves primarily with α-tocopherol, and serum or plasma has been the most frequently used form of sample. For the determination of α-tocopherol, both normal and reverse phase methods have been successfully used. This chapter presents a sensitive, rapid method suitable for the extraction and determination of tocopherols in a range of small-tissue samples.
TL;DR: This chapter examines the possibility of metabolic regulation by thiol/disulfide exchange with respect to each of the six criteria: the intracellular thiol / disulfide status should vary in vivo in response to some metabolic signal.
Abstract: Publisher Summary The process of thiol/disulfide exchange provides a mechanism for the equilibration of the sulfhydryl oxidation state of proteins with the thiol/ disulfide status of the surrounding environment. If this process occurs in vivo and results in changes in the activities of certain enzymes, changes in enzyme activity could be coupled to changes in the redox potential of the cell. This chapter examines the possibility of metabolic regulation by thiol/disulfide exchange with respect to each of the six criteria: (1) the intracellular thiol/disulfide status should vary in vivo in response to some metabolic signal; (2) the oxidation of protein sulfhydryl groups by thiol/disulfide exchange should activate some enzymes, inactivate others, and not affect the activities of others; (3) the thiol/disulfide redox potential of a regulated protein should be near the observed thiol/disulfide ratio in vivo ; (4) thiol/disulfide exchange reactions must be kinetically competent under physiological conditions; (5) for regulated enzymes, the oxidized and reduced forms of the enzyme should both be observable in vivo ; and (6) the response of particular enzyme activities to oxidation by thiol/ disulfide exchange should be consistent with the metabolic function of the enzyme.
TL;DR: A new high-performance liquid chromatography (HPLC) procedure for the analysis of free malonaldehyde in biological samples, such as cells and cell fractions and autoxidized fatty acids, originally developed for studying mal onaldehyde formation by rat liver microsomes is described.
Abstract: Publisher Summary Malonaldehyde is generally measured by the 2-thiobarbituric acid (TBA) method. The TBA method, however, is not specific for free malonaldehyde, because many other substances that can occur in biological material give positive reactions with TBA. The validity of equating the TBA reaction with the content of malonaldehyde itself in the sample, however, has been questioned many times. A new high-performance liquid chromatography (HPLC) procedure for the analysis of free malonaldehyde in biological samples, such as cells and cell fractions and autoxidized fatty acids, originally developed for studying malonaldehyde formation by rat liver microsomes, is described. Even though the determination of malonaldehyde by HPLC is rather quick and very sensitive, it should not be considered as a substitute for the common TBA assay when a large number of samples must be analyzed within a short time period. In such cases, HPLC analysis of a few selected samples will generally provide sufficient information about the existence or absence of free malonaldehyde and the quantitative relationship between the TBA number and the amount of free malonaldehyde.
TL;DR: Evidence has accumulated that low-level chemiluminescence in the near-infrared provides useful information on oxidative processes in cells and tissues and extrapolation from such measurements to other reactions must be made with due caution in each case.
Abstract: Publisher Summary Because of its high reactivity, 1 O 2 may be classed as one of the aggressive oxygen species known to arise in biological systems. Chemically, the reactions include addition to olefins (ene reactions) leading to allylic hydroperoxides, additions to diene systems leading to endoperoxides, and further types of reactions leading to dioxetanes or oxidation of certain heteroatoms. Photon counting has been applied to biological systems, and recently, evidence has accumulated that low-level chemiluminescence in the near-infrared provides useful information on oxidative processes in cells and tissues. Advantages of the technique are that it is noninvasive and provides continual monitoring. Major biological sources of 1 O 2 can be grouped into those systems involving the interaction of organic oxygen radicals, such as lipid peroxy radicals and that arise from lipid peroxidation, and those which involve reactions of reduced oxygen intermediates, such as O 2 · - , H 2 O 2 , HO·. A third group might be represented by those systems that probably involve an enzymatic activation of oxygen––that is, cyclo-oxygenase activity during prostaglandin biosynthesis. Photosensitization reactions represent a fourth category. 1 O 2 in general will arise in very low yield and must be considered as a side-product of major radical processes. This implies possible pitfalls in the interpretation of low-level chemiluminescence measurements, and extrapolation from such measurements to other reactions must be made with due caution in each case.
TL;DR: This chapter discusses the chemistry of the reduction products of dioxygen, but one should be aware of the marked influence of all of these factors on most reactions of dIOxygen.
Abstract: Publisher Summary The chemistry of molecular oxygen, or dioxygen as it is increasingly coming to be called, is dominated by the relative reluctance with which the element reacts with most, but not all, compounds. This quality is rarely thermodynamic in origin; rather the slow rate of reaction is associated with either the strong oxygen–oxygen bond or the character of the ground state of dioxygen or both. If a reaction is to take place with dioxygen in its ground state, there must be a change of spin at some stage during the reaction. This is forbidden, within the limitations of the descriptions used; at the very least, the reactions are improbable. For dioxygen to react rapidly, this spin restriction should be removed or the oxygen–oxygen bond should be partially weakened or both of these should occur simultaneously. It is not surprising that much of the chemistry of dioxygen is concerned with reactions with paramagnetic species, with electron-donating species, with light, or with various combinations of these three factors. This chapter discusses the chemistry of the reduction products of dioxygen, but one should be aware of the marked influence of all of these factors on most reactions of dioxygen. They are often, if not always, responsible for the promotion or catalysis of oxidation reactions.
TL;DR: This chapter describes the analysis of these secondary products forming the basis of several tests for peroxidation, and their assay offers the next most direct measure ofperoxidation.
Abstract: Publisher Summary The most direct and meaningful quantitation of peroxidation would be achieved from measurements of either the loss of substrate or the uptake of oxygen. Though these two measures have been used with considerable success by, for example, rubber chemists and food scientists, they are often not relevant in the biochemical context where the concept of a single, definable, substrate may not hold and where there are other oxygen-utilizing processes, such as respiration, that could mask any extra demand for oxygen for autoxidative degradation. The major initial reaction products of lipid peroxidation are hydroperoxides, and their assay offers the next most direct measure of peroxidation. These rather labile species can undergo both enzymic and nonenzymic decomposition to give products that include volatile hydrocarbons, malonaldehyde and malonaldehyde precursors, and carbon monoxide. This chapter describes the analysis of these secondary products forming the basis of several tests for peroxidation.
TL;DR: The action of PDI in catalyzing the interchange of inter- and intramolecular disulfides in scrambled ribonuclease results in the regain of the native disulfide pairing, native conformation, and concomitant return of rib onuclease activity against RNA.
Abstract: Publisher Summary Protein disulfide-isomerase (PDI) catalyzes the formation of native proteins from the reduced denatured state. When incubated in the presence of a thiol compound, PDI catalyzes the regain of native ribonuclease structure from the scrambled ribonuclease, with concomitant return of activity toward RNA. This assay is based on a patently nonphysiological substrate. It is very sensitive and has permitted the study of PDI activity in a number of contexts, making it possible to propose a physiological role for this activity. The chapter describes the preparation of scrambled ribonuclease from the beef pancreatic ribonuclease A, which contains a complex mixture of various molecular weight components. The substrate, scrambled ribonuclease, is essentially inactive in the hydrolytic cleavage of high-molecular-weight RNA, having about 2% of the activity of native ribonuclease. The action of PDI in catalyzing the interchange of inter- and intramolecular disulfides in scrambled ribonuclease results in the regain of the native disulfide pairing, native conformation, and concomitant return of ribonuclease activity against RNA. Thus, the activity of protein disulfide-isomerase is assayed by a time-course incubation during which aliquots are removed and ribonuclease activity toward RNA is measured. Protein disulfide-isomerase is very widely distributed and has been detected in most vertebrate tissues, although detailed studies have been confined to the enzyme from the liver.
TL;DR: This chapter focuses on bactericidal activity as measured by decrease in colony-forming units, using Escherichia coli as the target, MPO as the peroxidase, and chloride as the halide.
Abstract: Publisher Summary Peroxidases when combined with H 2 O 2 and a halide (chloride, bromide, iodide, and pseudohalide thiocyanate) form a potent cytotoxic system, which contributes to the host defense against invading microorganisms and possibly tumor cells. Neutrophils and monocytes contain the same peroxidase (myeloperoxidase, MPO), and eosinophils a different peroxidase (eosinophil peroxidase, EPO), in cytoplasmic granules, and these enzymes are discharged into the phagosome following particle ingestion. Phagocytosis also is associated with a respiratory burst and much of the added oxygen consumed is converted to H 2 O 2 . Peroxidase, H 2 O 2 , and a halide interact in the phagosome to destroy the ingested organism. The components of the peroxidase system can also be released extracellularly where they may attack adjacent normal or malignant cells, uningested organisms, or soluble mediators. A variety of methods has been employed for the measurement of the toxicity of the peroxidase system. These methods depend on the nature of the target cell and include the measurement of replication in growth medium, Cr release, metabolic activity, and morphologic changes. This chapter focuses on bactericidal activity as measured by decrease in colony-forming units, using Escherichia coli as the target, MPO as the peroxidase, and chloride as the halide.
TL;DR: It was proposed that the latter might be a useful extracellular parameter to assess hydroperoxide turnover in cells and tissues, and appropriate caution is required in each particular case in which the parameter is used.
Abstract: Publisher Summary Because of the release of glutathione disulfide (GSSG) into the extracellular space, cells, such as erythrocytes, or tissues, such as eye lens or liver, were found to respond to oxidizing conditions. Under steady-state conditions, the rate of metabolism of an externally added model hydroperoxide, t-butyl hydroperoxide, was observed to be linearly related to the rate of efflux of GSSG from the isolated perfused rat liver; in this organ, about 3% of the flux through glutathione (GSH) peroxidase is represented by an extracellular appearance of GSSG. Thus, even though most cell types contain an appreciable activity of GSSG reductase, a rise in the intracellular production of GSSG is accompanied by an export of the disulfide; apparently, there are multiple ways of disposing of GSSG, which may accumulate. Based on the relation between hydroperoxide metabolism and GSSG efflux, it was proposed that the latter might be a useful extracellular parameter to assess hydroperoxide turnover in cells and tissues. Because of the multiple factors that may influence the rate of GSSG efflux, appropriate caution is required in each particular case in which the parameter is used.
TL;DR: This chapter discusses those techniques and presents the major observations on protein disulfide formation in vivo as a post-translational modification using the techniques developed in vitro.
Abstract: Publisher Summary Protein disulfide formation in vivo as a post-translational modification is expected to depend upon both the conformation and the environment of the polypeptide chain during and after biosynthesis. Extrapolations from in vitro results may be informative, but the in vivo process should be amenable directly to study with the appropriate protein biosynthesis system, using the techniques that have been developed in vitro . This chapter discusses those techniques and presents the major observations. Disulfides do not form spontaneously among thiols, even when in close proximity, unless there is an appropriate electron acceptor (A) present. The most pertinent electron donor and acceptor to use would be the one that is involved in vivo . Glutathione (GSH) in its oxidized form, GSSG, is the most likely candidate owing to its ubiquity and relatively high concentrations in most organisms. Upon adding a disulfide reagent to a reduced protein, two sequential thiol-disulfide exchange reactions are required to form one protein disulfide. The first is a simple chemical reaction between the disulfide reagent and one cysteine (Cys) thiol group to generate the mixed disulfide. The second step is the one in which a second Cys thiol group reacts with the mixed disulfide to form the protein disulfide.
TL;DR: The free radical chemistry of lipid peroxidation is complex as discussed by the authors, and the classical mechanism of autoxidation involving a peroxy radical abstracting hydrogen atom from lipid and oxygen addition to the carbon radical thus formed must be modified to include (1) peroxide radical β fragmentation and (2) peroxyradical cyclization.
Abstract: The free radical chemistry of lipid peroxidation is complex The classical mechanism of autoxidation involving a peroxy radical abstracting hydrogen atom from lipid and oxygen addition to the carbon radical thus formed must be modified to include (1) peroxy radical β fragmentation and (2) peroxy radical cyclization A host of diene hydroperoxides, cyclic peroxides, bicyclic peroxides and epoxy alcohols may be formed in free fatty acid or phospholipid autoxidation The distribution of products and the effects of hydrogen atom donors on product distribution are understandable by referring to a general scheme for autoxidation described in Scheme III and in Ref 10
TL;DR: The chapter explores that mononuclear cell have a lower density than granulocytes and erythrocytes, and lymphocytes that overlap in density with monocytes appear to be more sensitive to an increase of osmolality than the monocytes.
Abstract: Publisher Summary This chapter reviews that cell separation techniques are based on differences in physical and/or biological properties of cells. The physical characteristics such as particle, density, and size determine the sedimentation rate in a liquid medium. Techniques for separation of the major subgroups of white blood cells are mostly based on distinct density differences. The chapter explores that mononuclear cell have a lower density than granulocytes and erythrocytes. Monocytes have a lower density than other cells in human blood and may be separated by density gradient centrifugation. It explains that the basis for using these principles for cell separation is that the various cell types have different osmotic sensitivities as a result of, for instance, different water content. The chapter reviews that in any event, the lymphocytes that overlap in density with monocytes appear to be more sensitive to an increase of osmolality than the monocytes. Thus with the correct combination of density and osmolality all of the cells loaded onto the gradient, except for the low-density fraction of the monocytes, move to the bottom during centrifugation. A monocyte fraction of 95-98% purity can then be collected from the top of the gradient layer.