A rapid whole-cell assay for superoxide dismutase

A procedure is described for the intact-cell assay of superoxide dismutase(s). The technique involves the use of toluene which renders the cells permeable to the necessary components of a photochemical assay for superoxide dismutase. Whole-cell superoxide dismutase activities from a number of procaryotic and eucaryotic microorganisms compare with cell-free activities and with activities reported in the literature. Using this procedure, changing levels of superoxide dismutase are readily monitored under conditions known to modulate superoxide dismutase activity assayed in vitro. In whole cells of Escherichia coli, exogenous methyl viologen causes a marked increase in superoxide dismutase activity, whereas in the cyanobacterium, Microcystis aeruginosa, such treatment leads to a marked, light-dependent loss of whole-cell superoxide dismutase activity.     

A picomolar spectrophotometric assay for superoxide dismutase

During the aerobic xanthine oxidase reaction, O₂^? is produced and accumulates to a steady state determined by a balance between the rate of production of this radical and its rate of dismutation. Addition of ferricytochrome c then results in a biphasic reduction, the very rapid phase of which reflects reaction of the accumulated O₂^?, while the slower phase corresponds to the continuing production of this radical. Superoxide dismutase suppresses the accumulation of O₂^? during the xanthine oxidase reaction and thus diminishes the burst of reduction seen upon addition of ferricytochrome c. This effect has been utilized, at pH 10.2, as the basis of an assay that permits measurement of picomolar levels of superoxide dismutase. The theory and practice of this ultrasensitive assay are described.     

Luminol assay for microdetermination of superoxide dismutase activity: its application to human fetal blood

A microtechnique for determining the superoxide dismutase activity in erythrocytes is described. This technique involves the inhibition of luminol-enhanced chemiluminescence of superoxide anion generated by xanthine-xanthine oxidase. Measurements required a steady-state chemiluminescence whether superoxide dismutase was present or absent; the level of luminescence was correlated to enzyme activity. Superoxide dismutase activity measured by this technique was 836 +/- 112 micrograms/g of hemoglobin for whole blood and 834 +/- 109 micrograms/g of hemoglobin for erythrocytes. When the reference technique was applied to larger amounts of blood, the results were 862 +/- 58 and 858 +/- 116 micrograms/g of hemoglobin for whole blood and washed erythrocytes, respectively. The enzymatic activity of superoxide dismutase from fetal blood (obtained by venipuncture in utero and of 19-26 weeks gestational age) was similar to that of adult blood, when measured by the new technique.     

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.     

NMR method for superoxide dismutase assay in brain and liver homogenates.

A method for copper- and manganese-containing superoxide dismutase (Cu- and MnSOD) assay in tissue homogenates such as liver and brain, based on the measurement of the longitudinal nuclear relaxation time (T1) of F-, has been developed as a preliminary approach to in vivo measurement of these enzymes. The relaxation rate of F-, which increases linearly with the SOD concentration, also depends on the oxidation state of the metal ion present in the active site of the enzyme. The relaxivity values of the oxidized, reduced and turnovering CuSOD were found to be 9.6 x 10(6), much less than 1 x 10(2) and 5.2 x 10(6) M-1 s-1, respectively, while for MnSOD the corresponding values were 2.9 x 10(6), 4.2 x 10(6) and 3.6 x 10(6) M-1 s-1, respectively. These high relaxivity values allow the detection of SODs in brain and liver homogenates where, under aerobic conditions, these enzymes appear in the steady-state. The contribution of the two types of SOD to the F- relaxation rate in the homogenates was measured by addition of either diethyldithiocarbamate or cyanide, both of which selectively inhibit the CuSOD. The comparison between NMR and activity data confirmed the possibility of carrying out accurate and precise measurements of SODs in homogenates by NMR.     

Superoxide--driven oxidation of quercetin and a simple sensitive assay for determination of superoxide dismutase

Oxidation of quercetin at pH 10 was shown to be a free radical chain reaction involving superoxide and hence inhibitable by superoxide dismutase (SOD) (EC 1.15.1.1). The degree of inhibition of quercetin oxidation was a function of SOD concentration, and fifty percent inhibition was produced by approximately 1.5 ng/ml of pure enzyme. This reaction proved to be a very useful tool for a rapid and highly sensitive measurement of SOD in crude tissue extracts and other biological samples.     

A critical reevaluation of some assay methods for superoxide dismutase activity

We have compared the direct method of pulse radiolysis to the indirect methods of cytochrome c and nitroblue tetrazolium for assaying the superoxide dismutase activity of a compound. We have shown that with pulse radiolysis, where high concentrations of O2- are generated, the "turnover" rate constant, kcat, can be determined directly, while with the indirect methods, where relatively low steady state concentrations of O2- are formed, the value of kcat determined by these methods, can be orders of magnitude lower than that determined directly. The main reason for the lower values obtained with the indirect methods is due to the fast reoxidation of the reduced compound by molecular oxygen. Additional problems which arise with the use of indirect methods for determining superoxide dismutase catalytic activity are discussed.     

Polarographic determination of superoxide dismutase.

A polarographic procedure is described which allows determination of the catalytic constants for superoxide dismutase-catalyzed reactions. The method presents a single and rapid evaluation of the enzyme concentrations as well as determination of its activity under different conditions; e.g., pH between 9 and 13, presence of urea, guanidine, sodium dodecyl sulphate and inhibitors such as CN^? and N₃^?.The results fit very well with data previously obtained with other methods and show that this polarographic procedure can be used under conditions that render the other methods unsuitable for the measurement of the enzyme activity.     

纯化超氧化物歧化酶的新工艺

为探索简便实用纯化ＳＯＤ的工艺路线,以人或猪血红细胞溶血上清液,经铜胺中空纤维透析器（分子量截留值为１５ｋＤ）透析和超滤,收集分子量大于１５．０ｋＤ的物质,再加热６０℃１０ｍｉｎ,离心取上清即得。Ｃｕ、ＺｎＳＯＤ和ＭｎＳＯＤ分子量分别为３２．０ｋＤ和８０．０ｋＤ。人血和猪血纯化的ＳＯＤ总收率分别为８８．２％和８９．２％,比活性分别为１７４２９Ｕ／ｍｇ和１８２２８Ｕ／ｍｇ。工艺简便实用,适于工业纯化生产。     

超氧化物歧化酶在临床上的应用

对超氧化物歧化酶(SOD)、重组人SOD(rh-SOD)在临床上的应用进行了概括和介绍。动物来源的SOD目前主要应用于治疗肿瘤放疗的后遗症、各种炎症以及多种皮肤病,rh-SOD则较多应用于心脏、肾脏等器官的保护和移植过程以及心血管疾病的治疗。     

A spectrophotometric assay for superoxide dismutase activities in crude tissue fractions.

A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.     

The pyrogallol assay for superoxide dismutase: Absence of a glutathione artifact

Reduced glutathione was found to affect the assay for superoxide dismutase when autooxidation of epinephrine, but not pyrogallol, was used as the indicator. Glutathione concentrations in the micromolar range, which correspond to levels in erythrocyte extracts, were capable of perturbing the epinephrine assay method and causing overestimation of enzyme content. The pyrogallol method was not significantly affected by large excesses of glutathione and appears to be a superior method for tissue extracts likely to be rich in glutathione.     

A sensitive assay for superoxide dismutase based on the autoxidation of 6-hydroxydopamine

A simple, sensitive spectrophotometric assay system for superoxide dismutase (SOD) has been developed. This assay is based on the inhibitory effects of SOD on the initial rate of 6-hydroxydopamine autoxidation. The inhibition of 6-hydroxydopamine autoxidation was virtually linear to an SOD concentration of approximately 100 ng of SOD/ml (about a 50% inhibition at 100 ng/ml; there was a greater inhibition at higher SOD concentrations). With this assay system it was determined that SOD levels in rat brain, liver, and spinal cord were 84, 660, and 56 μg of SOD/g of tissue, respectively. These results agree very well with results obtained by other assays.     

Enzymatic and non-enzymatic assay of superoxide dismutase.

Superoxide dismutase from breef brain and rat liver was assayed in an enzymatic system, using xanthine oxidase, and a non-enzymatic system, based on aerobic reduction of nitro-blue tetrazolium in presence of phenazine methosulphate. The non-enzymatic assay is rapid and simple and permits simulatneous analysis of many samples. Similar results are found by the two methods of assay of superoxide dismutase.     

Quantitative histochemical assay for superoxide dismutase in rat brain.

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.     

An assay for superoxide dismutase activity in mammalian tissue homogenates

During the course of measuring superoxide dismutase (SOD) activity in rat breast tissue, interferences in the nitroblue tetrazolium (NBT) and cytochrome c assay systems were noted. These interferences inhibit accurate measurement of SOD activity in breast tissues, necessitating the development of a new NBT-based assay that includes compounds capable of inhibiting tissue specific interferences. The most effective compounds were metal chelators that were also electron transport chain inhibitors. Bathocuproine sulfonate (BCS) was the most effective of these compounds. The inclusion of BCS in the NBT assay system was shown to make the accurate measurement of SOD activity in tissues with interferences possible.     

Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase.

Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of autoxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, indicating the participation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide dismutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.