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.     

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.     

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.     

An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation

Superoxide dismutase activity was assayed in terms of its ability to inhibit the radical-mediated chain-propagating autoxidation of epinephrine. The enzyme assay based on adrenochrome absorption at 480 nm has been improved by measuring the absorption change at 320 nm. This alternative procedure was found to be 6 to 10 times more sensitive and more consistent than that measured at 480 nm.     

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.     

Reevaluation of assay methods and establishment of kit for superoxide dismutase activity

Superoxide dismutase (SOD) activity was measured by seven assay methods. The nitrite method was found to be the best for our SOD assay kit. This method was then modified to give better sensitivity and minimize interference by coexisting protein, a factor which has been previously ignored. Hydroxylamine or its O-sulfonic acid, xanthine oxidase, hypoxanthine, EDTA, and the sample were incubated with or without KCN at pH 8.2, 37°C, for 30 min. Diazo dye-forming reagent was added and the absorption was measured at 550 nm. Human plasma and erythrocyte lysate from healthy adults and Down's syndrome patients were assayed by this SOD kit and by the cytochrome c method. Our kit gave 8.5 times higher sensitivity than the cytochrome c method. This high sensitivity allowed the use of a simple spectrophotometer and, moreover, only one dilution was needed to determine the SOD unit with the help of our formulas. Good recovery, reproducibility, and stability of reagents were demonstrated.     

An assay for the detection of superoxide dismutase in individual Escherichia coli colonies

A method for detecting superoxide dismutase activity in individual colonies of Escherichia coli was developed. The assay involves the lysis of individual cells in colonies on filter papers by a series of lysozyme, chloroform, and freeze-thaw treatments. Filters are placed on agar plates to allow diffusion of cellular enzymes into a solid matrix. A nitroblue tetrazolium overlay is applied to detect superoxide dismutase activity. Colonies possessing activity produce achromatic zones against a dark Formazan background. The assay can detect the presence of superoxide dismutase and the relative amount of enzyme as well. This assay provides a method for screening a population of cells for mutants deficient in or overproducing superoxide dismutase.     

Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species.

The contents of extracellular superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase were determined in tissues from nine mammalian species. The pattern of CuZn superoxide dismutase distribution was similar in all species, with high activity in metabolically active organs such as liver and kidney and low activity in, for example, skeletal muscle. Mn superoxide dismutase activity was high in organs with high respiration, such as liver, kidney, and myocardium. Overall the Mn superoxide dismutase activity in organs was almost as high as the CuZn superoxide dismutase activity. The content of extracellular superoxide dismutase was, almost without exception, lower than the content of the other isoenzymes. The pattern of tissue distribution was distinctly different from those of CuZn superoxide dismutase and Mn superoxide dismutase. The tissue distribution of extracellular superoxide dismutase differed among species, but in general there was much in lungs and kidneys and little in skeletal muscle. In man, pig, sheep, cow, rabbit and mouse the overall tissue extracellular superoxide dismutase activities were similar to each other, whereas dog, cat and rat tissues contained distinctly less. There was no general correlation between the tissue extracellular superoxide dismutase activity of any of the various species and the variable plasma activity. The ratio between the plasma and the overall tissue activities was high, for some species over unity, providing further evidence for the notion that one role of extracellular superoxide dismutase is as a plasma protein.     

An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.     

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.     

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 sarcoplasmic reticulum Ca2(+)-ATPase activity in muscle homogenates

A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca2(+)-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established that give maximal SR Ca2(+)-ATPase activity, while eliminating Ca2(+)-dependent myofibrillar ATPase activity and reducing Ca2(+)-independent or background ATPase activity. High [Ca2+] (20 mM) could be used to selectively inhibit the SR Ca2+ ATPase. Identification of the Ca2(+)-dependent ATPase activity in muscle homogenates as being SR Ca2+ ATPase was based on a comparison of several parameters using homogenate material and purified SR. The following parameters were compared and found to be the same in homogenate and SR: activation and inactivation between 0 and 20 mM Ca2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca2+ ATPase. The method is illustrated with the analysis of homogenates prepared from freeze-dried muscle fibers and thin sections of muscles typically used in microscope analyses as well as an analysis of freshly prepared homogenates from various types of muscle, which shows a good correlation over a wide range between SR specific Ca2(+)-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 micrograms of tissue (wet weight) in a volume of 25 microliters.     

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.     

An assay of collagenase activity using enzyme-linked immunosorbent assay for mammalian collagenase

A new method was developed for the measurement of collagenase activity using the enzyme-linked immunosorbent assay (ELISA). Rabbit colon wall collagenase, pepsin-soluble rat skin type I collagen, and its antisera were used in the present experiment. After the collagenase-degraded portion of the collagen coated on the microwell was released, the immunoreaction of the residual collagen on the microwell to anticollagen sera was determined by ELISA. This method was approximately 10 times more sensitive than the conventional assay procedure using [14C]-glycine-labeled reconstituted collagen fibrils as substrate. It was suitable for screening a large number of samples without radioisotopes.     

Antioxidant superoxide dismutase activity in obese children

The aim of the study was to evaluate the antioxidative Cu/Zn-SOD (superoxide dismutase) response to obesity-related stress in obese children compared to a similar-aged control group. Forty-eight exogenic obese children and 11 healthy children were compared for red cell Cu/Zn-SOD, glucose, and lipid profiles and the relations between the were investigated. Antioxidant response as Cu/Zn-SOD was significantly higher in the obese group (p<0.05). Although glucose and lipid levels were statistically higher in the obese group, a certain relation with the SOD level was not established in childhood. This is the first study showing the oxidative stress caused by obesity and related antioxidative response even in the childhood period. Interventions, including diet modifications, should be kept in mind to diminish the obesity-related oxidative stress from the childhood period.     

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.     

A flash-photometric method for determination of reactivity of superoxide: application to superoxide dismutase assay

Pulse-generation of O2- by a flash was used to determine the reactivity of O2-, O2- was produced within 10 ms by a flash of light through the excitation of FMN in the presence of N,N,N',N'-tetramethylethylenediamine and oxygen. Kinetic analysis of cytochrome c reduction by O2- generated by flash yielded the reaction rate constant between cytochrome c and O2- and the spontaneous disproportionation rate constant of O2-. We applied it for superoxide dismutase assay using a linear relation between superoxide dismutase concentration and the apparent rate constant of cytochrome c reduction by O2-. The catalytic rate constant and activation energy at pH 7.3 of bovine liver Cu,Zn-superoxide dismutase were found to be 1.75 x 10(9) M-1 . s-1 at 25 degrees C and 26.9 kJ . M-1, respectively. The kinetics of O2- decay can be also monitored at 240 nm in this flash-photometric system and gave the spontaneous disproportionation rate constant of O2- and the catalytic rate constant of superoxide dismutase.     

Temperature dependence of superoxide dismutase activity in plankton

The effect of temperature (from 1 to 37 °C) on in vitro effective superoxide dismutase (SOD) activity of several organisms was investigated and compared. Antarctic plankton, cultures of the alga Nannochloropsis sp., and the cyanobacterium Synechococcus strain WH 7803, and pure bovine erythrocyte SOD was studied. It was found that in all cases SOD activity increased with decreasing temperature within the temperature range assayed, in the Polar as well as the temperate plankton cells. This behavior of SOD is counterintuitive in terms of our experience when looking at enzyme activity or any other chemical reaction. We suggest a theoretical explanation for this apparently odd behavior. The advantage of such behavior is that the same amount of antioxidant will act better under low temperatures when reactive oxygen species (ROS) increase. Moreover, this protective process would act in vivo at a faster pace than the ex novo enzyme synthesis.