can be used as a positive assay for SOD activity in which the autoxidation rate is stimulated in proportion to the level of added SOD. The positive method is an order of magnitude less sensitive than the negative assay, but it may be applied in situations in which the SOD concentration is high, such as during purification of SOD. Acknowledgements This work was supported by Public Health Service Research Grant AI-19695 from the National Institute of Allergyand InfectiousDiseases;Grant C-900from the RobertA. Welch Foundation; and a grant from the American Heart Association, Texas Affiliate.

[20] A s s a y for S u p e r o x i d e D i s m u t a s e B a s e d o n Chemiluminescence of Luciferin Analog By MINORU NAKANO

Principle The methods for estimating superoxide dismutase (SOD) concentrations in erythrocytes and tissues are based on the inhibitory effect of SOD extracted from these materials. Using 2-methyl-6-(p-methoxyphenyl)-3,7dihydroimidazo[1,2-a]pyrazin-3-one (MCLA), the chemiluminescence induced by a hypoxanthine-xanthine oxidase system (an O2--generating system) is measured and the inhibition determined. Preparation of 2 -Methyl-6 -(p -methoxyphenyl)-3, 7 -dihydroimidazo[ 1,2 -a ] pyrazin-3-one Hydrochloride MCLA can be synthesized from 2-amino-5-(p-methoxyphenyl) pyrazine according to the following procedure, l To a solution of 2-amino5-(p-methoxyphenyl)pyrazine (100 mg) and 40% aqueous methylglyoxal (0.16 ml) in methanol (4.1 ml) is added concentrated hydrochloric acid (0.1 ml). The mixture is heated and stirred under an atmosphere of nitrogen at 60-70 ° for 1.5 hr. After drying in vacuo, the product mixture is partially purified by silica gel chromatography. The crude product which is eluted from the column in 2-propanol is recrystallized from benzenemethanol to give a pale yellow crystalline power (89 mg, 70%), mp 197201 ° (dec.). Analysis: Calc. for CI4H1402N3CI: C, 57.64; H, 4.84; N, I A. Nishida, H. Kimura, M. Nakano, and T. Goto, Clin. Chim. Acta 179, 177 (1989).


Copyright © 1990by Academic Press, lnc. All rights of reproduction in any form reserved.




14.40%. Found: C, 57.62; H, 4.70; N, 14.39%. UVmax (MeOH), nm (8): 430 (8260), 357 (5460), 261 (20800), 203 (20900); (MeOH/HCI): 348 (5080), 278 (21700), 208 (18600); (MeOH/NaOH): 409 (6000), 348 (5530), 264 (22900), 207 (20900). Mass spec. (EI): m/z 255 (M+), 240,226, 186, 171.1H NMR ( D M S O - d 6 + D 2 0 ) , 8:8.05 and 7.70 (each IH, s), 7.69-7.73 (2H, A E ' X 2 ' , J = 9 Hz), 7.04-7.11 (2H, A E ' X 2 ' , J = 9 Hz), 3.82 (3H, s), 2.36 (3H, s). The e430nm value of 9600 M-lcm -1 in water can be used for preparation of the MCLA solution.

Preparation of SOD Samples

Preparation ofErythrocyte Lysates. Blood is drawn into heparinized syringes (20 units/ml) from human volunteers or animals. Samples are prepared according to Oyanagui. 2 Heparinized blood (1-3 rnl) is centrifuged at 2500 rpm for 30 min at 0-4 ° and the plasma carefully separated. Saline is added to the erythrocyte pellet up to the original volume, and 0.4 ml of the cell suspension is transferred to a 50-ml centrifuge tube. After washing the erythrocytes twice with saline, the cells are diluted with water to 4 ml to lyse the erythrocytes. Ethanol (1 ml) and chloroform (0.6 ml) are then added to remove hemoglobin (Tsuchihashi method3). The tubes are shaken vigorously for 15 min and centrifuged at 2500 rpm for 10 min at 0-4 °. Exactly 0.1 ml of the water-ethanol layer is aspirated and diluted with 0.7 ml of water to make a final concentration of 1/1000 of whole blood cells and 0.25% ethanol. Ethanol (2.5%) is used for further dilution of erythrocyte lysates to obtain a 0.25% solution. Granulocytes and Lymphocytes from Human Blood. Granulocytes and lymphocytes are obtained by the methods described by Boyum. 4 Granulocytes and lymphocytes are over 95% homogeneous. Washed granulocytes obtained from 1 ml whole blood or washed lymphocytes obtained from 3 ml whole blood are resuspended in 1 ml of Hanks' balanced salt solution and are sonicated over ice in 30 consecutive 0.5-sec bursts at a power setting of 30 W (Branson sonifier). The sonication is repeated after 1 min. The sonicated sample is centrifuged at 78,000 g for 60 min, and the clear supernatant is used for the assay of total SOD. An aliquot (0.4 ml) of the supernatant is further treated with sodium dodecyl sulfate (SDS) to inactivate Mn-SOD activity (see below). Tissues from Rats. Rats, weighing between 230 and 250 g, are anesthetized with ketamine (60 mg/kg) and subjected to a gradual perfusion with 2 y. Oyanagui, Anal. Biochem. 142, 290 (1984). 3 M. Tsuchihashi, Biochem. Z. 140, 65 (1923). 4 A. Boyun, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77 (1967).




lactate-Ringer solution (pH 7.0) containing heparin (20 units/ml), allowing outflow from the decending aorta through femoral vein, and sacrificed by decapitation. Each organ is immediately removed, weighed, and homogenized in 9 volumes (w/v) of buffered sucrose solution (0.25 M sucrose in 20 mM Tris-HCl buffer containing 1 mM EDTA at pH 7.4). The homogenate is then sonicated over ice in 30 consecutive 0.5-sec bursts at a power setting of 60 W (Branson sonifier). This procedure is repeated 4 times at 1-min intervals. The sonicated sample is centrifuged at 78,000 g for 30 min, and the supernatant is diluted with 50 mM Tris-HCl buffer at pH 7.4 to achieve a 1000- to 5000-fold dilution of the wet tissue. An aliquot of the sample is further treated with SDS to inactivate Mn-SOD. Differentiation of Cu,Zn-SOD from Mn-SOD. Several methods have been used to distinguish Cu,Zn-SOD activity from Mn-SOD activity in whole tissues or blood cell extracts. These are the Tsuchihashi reagent, N,N'-diethyldithiocarbamate, sodium dodecyl sulfate (SDS), and CN-. Only the SDS method proved workable with MCLA chemiluminescence. Inactivation of Mn-SOD activity in the sample by SDS is accomplished as described by Bruce and Dennis. 5 SDS is added to the diluted tissue extract or to a mixture of bovine erythrocyte Cu,Zn-SOD and human liver Mn-SOD in 50-100 m M Tris-HCl buffer at pH 7.4 to obtain a final concentration of 2% (w/v), and the mixture is incubated at 37° for 2 hr to inactivate Mn-SOD. At the end of the incubation, the solution is cooled to 4° and mixed with 1/10 volume of 3 M KC1 to precipitate the SDS. After standing for 30 min at 4°, the mixture is centrifuged at 20,000 g for 10 min at 4 °. The supernatant obtained is used to assay for Cu,Zn-SOD. Assay of Xanthine Oxidase Activity Enzyme activity can be determined in glycine-HCl buffer at pH 8.8 and 37°, using hypoxanthine as substrate.6 One unit of enzyme is defined as the amount of the enzyme that catalyzes an increase in absorbance of 0.001/30 rain at 290 nm.

Reagents 30 ~ M MCLA in water (prepared from a stock solution of 300 ~ M MCLA) 1.5 mM hypoxanthine in water Xanthine oxidase (grade III, buttermilk, Sigma Chemical Co., St. Louis, MO) 5 L. D. Bruce and R. W. Dennis, Anal. Biochem. 123, 86 (1983). 6 G. G. Roussos, this series, Vol. 12A, p. 5.




Superoxide dismutase, bovine erythrocyte SOD (3500 units/mg of protein) 0.3 M Tris-HC1 buffer containing 0.6 m M EDTA at pH 7.8 Procedure. A standard reaction mixture contains 20 /~1 of MCLA, 100/xl of hypoxanthine, 50/zl of 6.5 units of xanthine oxidase, 6-200/zl purified bovine erythrocyte SOD or sample (or none), and 500/zl of the Tris-HC1 buffer containing EDTA and water, for a total volume of 3.0 ml. The chemiluminescence measurement is initiated by the addition of MCLA to the standard incubation mixture excluding xanthine oxidase, continued for 4 min without additions, and then continued for an additional 4 min after the addition of xanthine oxidase. Chemiluminescence can be measured with a Luminescence Reader (Aloka, BLR 102) at 25 ° and expressed as counts per minute. Preparation of the standard curve for MCLA in the standard incubation mixture excluding xanthine oxidase and SOD (control system) is based on the light emitted in the visible region (hmax 465 nm). Such luminescence originates from the autooxidation of MCLA and is a nonspecific chemiluminescence. The addition of xanthine oxidase to the above mixture (experimental system) rapidly enhances the luminescence, which reaches a maximum and then remains constant for at least 3 min. The luminescence in the system containing xanthine oxidase decreases with increasing SOD concentration. However, nonspecific luminescence is almost constant for 10 min after the addition of MCLA and is not significantly influenced by SOD. The xanthine oxidase-induced luminescence (I0), expressed in terms of light intensity (counts/min), can be calculated by subtraction of the nonspecific light intensity at 8 min (after the addition of MCLA) from the light intensity at 2 min (after the addition of xanthine oxidase). The same incubation experiments, except that SOD is present both in the experimental and the control systems, are carried out, and the enzyme-induced luminescence (Ii) is calculated in the same manner as described above. The percentage of SOD inhibition dependent on the xanthine oxidaseinduced luminescence (I0) can be calculated from Eq. (1) and is then plotted against the SOD concentration to obtain the standard curve. % Inhibition = (Io/Ii)/Io × 100


The SOD concentration for 50% inhibition of the xanthine oxidaseinduced luminescence has been compared with that obtained by the cytochrome c method. 7 The luminescence method may give 95 times higher sensitivity than the cytochrome c method. 7 j. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969).






Total SOD



Mn-SOD/Total SOD

Erythrocyte Granulocyte Lymphocyte

1.43 -+ 0.11 1.22 -+ 0.12 5.66 --- 0.42

1.43 -+ 0.11 1.09 -+ 0.16 4.57 - 0.53

0 0.13 - 0.05 1.10 -+ 0.31

0 10.7 -+ 5.1% 19.5 - 5.5%

Data are expressed as ng/10 5 cells and are means -+ standard deviations of five observations on samples measured in duplicate. T A B L E II SOD CONCENTRATIONS IN RAT TISSUESa

Tissue Brain Lung Heart Kidney Liver

Total SOD 133.4 115.6 191.2 516.0 819.2

-+ -+ -+ -----

31.0 23.8 32.0 43.2 113.2

Cu,Zn-SOD 94.4 84.8 106.2 413.0 603.2

-+ -+ -

16.5 10.2 20.8 60.5 158.8

Mn-SOD 39.0 30,8 85,0 103,0 216.0

-+ 26.0 - 16.7 -+ 18.6 --+ 53.2 - 51.0

Mn-SOD/Total SOD 27.7 25.6 44.5 19.1 27.4

+ -+ +

14.0% 8.6% 7.0% 10.2% 9.9%

Data are expressed as/zg/g wet weight and are means -+ standard deviations of five observations of samples measured in duplicate.

Assay of SOD in Sample The standard reaction mixture is used for the assay of SOD in the sample. Cu,Zn-SOD and Mn-SOD activities in human blood cells and rat tissues determined by the MCLA method are shown in Tables I and II. All SOD activities are expressed as nanograms (ng) or micrograms (/~g) of bovine erythrocyte SOD based on a specific activity of 3500 units/mg protein. The values for human blood (Cu,Zn-SOD activities in erythrocyte lysate) have been quoted from the previous report. 8 Cautions Regarding the Assay The xanthine oxidase-induced luminescence is inhibited by purified bovine serum albumin, s probably also by purified human albumin or other protein, at concentrations of more than 5/zg/ml of the reaction mixture. Protein concentrations in samples used for the assay are less than 1.2/xg/ml, which would not interfere with the assay of SOD by the lumi8 H. Kimura and M. Nakano, FEBS Lett. 239, 347 (1988).



[2 1]

nescence method. Glutathione, ascorbate, uric acid, and glucose are also inhibitory of the XOD-induced luminescence at concentrations greater than 4 x 10 -6, 3 × 10-8, 1 × 10-5, and 6 x 1024 M, respectively. However, the concentration of each inhibitor in the reaction mixture corresponds to more than 1 100-fold dilution of the concentration which interferes with the SOD assay. Thus, it is not necessary in the SOD assay to consider the interference by contaminants in the biological samples, provided they are adequately diluted. MCLA in solution is relatively unstable and autoxidizes with light emission at 465 nm. The stock solution of 300/xM MCLA in water (1 ml) should be stored at - 8 0 ° . The solution is then thawed, diluted with water to obtain 15 ~ M MCLA, and should be used as soon as possible thereafter.

[21] A u t o m a t e d A s s a y o f S u p e r o x i d e D i s m u t a s e in B l o o d

By MARY R. L'ABBI~ and PETER W. F. FlSCtaER The analysis of copper zinc superoxide dismutase (Cu,Zn-SOD) in erythrocytes and extracellular SOD in plasma must be carried out by an indirect method since the substrate (02 ~) is an unstable free radical. The O2-: is generated by xanthine plus xanthine oxidase. The SOD activity is calculated from the rate at which the generated 02 ~ reduces cytochrome c, a reaction which is followed spectrophotometrically. If the activity is high, more of the generated radicals are dismutated and less cytochrome c is reduced, whereas if the activity is low, the opposite is true. 1 The method is adapted to an Abbott VP Super System bichromatic analyzer, and, since it uses small volumes and allows for a relatively large number of samples, it is suitable for use in both clinical laboratories and research laboratories utilizing small animals. 2

Apparatus Abbott VP Super System bichromatic discrete analyzer (Abbott Laboratories Ltd., Diagnostics Division, Mississauga, Ontario, Canada) equipped with a 415/450 nm filter. The Abbott ABA-200 bichromatic analyzer has also been used. 2 1 j. M. M c C o r d and I. Fridovich, J. Biol. Chem. 224, 6049 (1969). 2 M. R. L ' A b b 6 and P. W. F. Fischer, Clin. Biochem. 19, 175 (1986).


Copyright © 1990by AcademicPress, Inc. All rights of reproductionin any form reserved.

Assay for superoxide dismutase based on chemiluminescence of luciferin analog.

[20] ASSAY FOR S O D WITH LUCIFERIN ANALOG 227 can be used as a positive assay for SOD activity in which the autoxidation rate is stimulated in pro...
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