ANALYTICAL BIOCHEMISTRY 70, 6 1 6 - - 6 2 0 (1976)

Inhibition of Nitrite Formation from Hydroxylammoniumchloride: A Simple Assay for Superoxide Dismutase Superoxide Dismutase, an enzyme which catalyzes the dismutation of the superoxide free radical ion (05-) into H~Oz and O5 (l) seems to be obligatory in aerobic organisms for the defense against oxygen toxicity (2,3). Based on the inhibition of O2--driven reactions, several systems for testing superoxide dismutase activities have been described, including (i) the reduction of cytochrome c or tetranitromethane (1); (ii) the oxidation of epinephrine (1,4); (iii) the reduction of nitrotetrazolium blue (5); (iv) the autoxidation of pyrogallol (6). We wish to report on another method for the determination of superoxide dismutase which is based on the inhibition of nitrite formation from hydroxylamine in the presence of O2:-generators (7,8). This method combines certain individual advantages of the four above mentioned assays: (a) a great number of samples can be assayed simultaneously; (b) the base-line-rate of nitrite formation in the absence of an O2--generating activity is low at physiological pH-values (pH 7.8-8.5); (c) the test system is inexpensive, sensitive, and specific for the superoxide free radical ion and for superoxide dismutase. Reagents

The following reagents are required for the test system. 1. For nitrite determination. (I) a-naphthylamine-solution: Dissolve 0.5 g ot-naphthylamine in 100 ml boiling distilled water, allow to cool to room temperature, and add 125 ml of glacial acetic acid. Fill up with 275 ml of cold distilled water to yield 500 ml. Store in the dark (aluminum foil). (I I) Sulfanilic acid solution: Dissolve 1.65 g of sulfanilic acid in 375 ml of warm distilled water, and add 125 ml of glacial acetic acid. 2. For hydroxylammoniumchloride oxidation. (I) Xanthine solution: Dissolve 11.42 mg of xanthine (Boehringer, Mannheim FRG) in 2.5 ml of 0.1 N NaOH; add 7.5 ml of distilled water. (II) Xanthine oxidase (Boehringer, Mannheim FRG, suspensions of 20 mg enzyme in 2 ml): Dilute 0.1 ml of enzyme suspension with 4.9 ml of ice-cold distilled water. (III) Hydroxylammoniumchloride solution (1/xmol/0.1 ml): Dissolve 6.9 mg of hydroxylammonium chloride (Merck, Darmstadt FRG) in 10 ml of distilled water. (IV) Phosphate buffer 65 mM, pH 7.8.

616 Copyright © 1976byAcademic Press, Inc. All rights of reproduction in any form reserved.

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Experimental Procedure Nitrite formation from hydroxylammoniumchloride is determined under the following conditions. (I) Incubation mixture: 2 ml total volume; distilled water, 0.5 ml; phosphate buffer (reagent 2/IV) 1.0 ml (65/xmol); xanthine oxidase (reagent 2/II) 0.3 ml (with 60 txg protein); xanthine (reagent 2/I) 0.1 ml (1.5/.~mol); hydroxylammoniumchloride (reagent 2/III) 0.1 ml (1 /~mol). (II) The reaction is started by the addition of xanthine oxidase. (III) The reaction is conducted at 25°C (water bath) for 20 rain. (IV) Determination of nitrite as the product of hydroxylammoniumchioride oxidation: Take 0.5 ml from the above reaction mixture and add to 0.5 ml sulfanilic acid solution (reagent 1/I I). Add 0.5 ml ~-naphthylamine solution (reagent l/I) and shake. After standing at room temperature for 20 min, the optical density of the mixture is determined at 530 nm in the spectrophotometer.

Results and Discussion Addition of superoxide dismutase to the incubation mixture described under Experimental Conditions yields an inhibition of nitrite formation. If a superoxide dismutase preparation from dried green peas (isolated after a modified procedure (9), according to Sawada et al., 1972, Ref. 10) is standardized with the cytochrome c test according to McCord and Fridovich (1), approximately 1/10 activity unit yields a 50% inhibition of hydroxylammoniumchloride oxidation. This inhibition of nitrite formation by green pea superoxide dismutase is reversed in the presence of KCN, an inhibitor of the copper-zinc-containing superoxide-dismutase (11), as shown in Table 1. As demonstrated in Table 1, two units of green pea-superoxide dismutase yield a complete inhibition of the Q--driven nitrite formation from hydroxylammoniumchloride if the extinction of the blank (E530 = 0.02 in the absence of xanthine oxidase) is subtracted. Superoxide dismutase from the blue green alga Spirulina platensis (prepared after a procedure described by Lumsden and Hall, Ref. 12) also inhibits hydroxylamine oxidation under the described conditions; this inhibition, however, is not reversed in the presence of 5 × 10-4M KCN (Table 2). The result shown in table 2 is in agreement with observations made with the nitrotetrazolium blue method (12). The hydroxylammoniumchloride method also allows one to differentiate between iron- or manganese-containing superoxide dismutases (no inhibition by cyanide) and copper-zinc enzymes which are not active in the presence of KCN. A differentiation between the two different types of superoxide dismutase (cyanide-sensitive and cyanide-insensitive) is not observed in dialyzed crude extracts of green peas, pig liver, or Spirulina platensis, since inhibition ofhydroxylammoniumchloride oxidation in presence of the

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EFFECT OF GREEN PEA-SUPEROXIDE DISMUTASE ON HYDROXYLAMMONIUMCHLORIDE OXIDATION IN THE PRESENCE AND ABSENCE OF 5 X 10-4 M K C N

Complete system Minus xanthineoxidase Complete system

SOD added (activity units a according to Ref. 1)

Extinction at 530 nm -KCN

+KCN

---

0.22 0.02

0.24 0.01

0.05 0.1 0.2 0.4 0.8 1.0 2.0 5.0

0.13 0.11 0.09 0.06 0.04 0.03 0.02 0.02

0.24 0.25 0.25 0.23 0.23 0.22 0.21 0.21

Percentage inhibition -KCN

+KCN

41 50 59 73 82 86 91 91

0 0 0 0 0 8 12 12

a One activity-unit contained ca. 2/~g of protein.

above preparations cannot be reversed by KCN. This effect is probably due to the presence of the cyanide-insensitive mitochondrial mangano enzymes (13). Dialysis of crude extracts is necessary in order to avoid possible superimposed effects of radical scavengers (like ascorbate) or electron donor systems, which eventually might obscure the results. TABLE 2 EFFECT OF SPIRULINA-SUPEROXIDE DISMUTASE ON HYDROXYLAMMONIUMCHLORIDE OXIDATION IN THE PRESENCE AND ABSENCE OF 5 >( 10-4 M K C N

Complete system Minus xanthineoxidase

SOD added (activity unitsa according to Ref. 1)

Extinction at 530 nm -KCN

---

0.18 0.00

0.1 0.2 0.4 0.8 1.2 1.8 2.0 4.0 10.0

0.11 0.07 0.06 0.03 0.02 0.02 0.01 0.00 0.00

a One activity-unit contained ca. 2.25/zg of protein.

+KCN

0.12 0.11 0.08 0.06 0.02 0.03 0.02 0.01 0.00

Percentage inhibition -KCN

39 61 67 83 89 89 95 100 100

+KCN

33 39 56 67 72 83 89 95 100

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INHIBITION OF HYDROXYLAMMONIUMCHLORIDE OXIDATION IN THE PRESENCE OF CRUDE EXTRACTS FROM GREEN PEAS, PIG LIVER, OR Spirulina Platensis IN THE PRESENCE AND ABSENCE OF K C N Extinction at 530 nm Dialyzed crude extract added to the complete system (ml) None 0.001 0.003 0.006 0.01 0.02 0.05

Crude extract from Green peas" KCN 0.24 0.19 0.09 0.06 0.04 0.02 0.02

+KCN

-KCN

+KCN

Bovine serum albumine a (10 mg/ml undialyzed)

0.28 0.12 0.07 0.05 0.05 0.02 0.02

0.21 0. l 0.07 0.04 0.03 0.03 --

0.28 0.16 0.07 0.06 0.05 0.03 --

0.21 0.20 0.19 0.20 0.20 0.20 0.21

Pig liver b

+KCN 0.34 0.15 0.11 0.08 0.05 0.03 0.03

KCN 0.21 0.07 0,05 0.05 0,04 0.04 --

Spirulina platensis c

a One hundred grams of soaked green peas were homogenized (2 min Sorvall Omni-Mixer) in 100 ml of 65 mM phosphate buffer, pH 7.8, and centrifuged for 30 min at 20,000g; 1 ml of dialyzed crude extract contained 18 mg of protein after recentrifugation (see above); protein was determined according to (14). b One hundred twenty-five grams of fresh pig liver were homogenized (2 rain Sorvall Omni-Mixer) in 50 ml of 65 mM phosphate buffer, pH 7.8, and centrifuged for 30 rain at 20,000g; 1 ml of dialyzed crude extract contained 118 mg of protein after recentrifugation. c Obtained by sonication (Branson sonifier, fullscale, 3 rain) of ca. 100 g of Spirulina platensis (fresh weight) and centrifuged for 30 min at 20,000g; 1 ml of dialyzed crude extract contained 31 mg of protein. a Obtained from Serva-Heidelberg.

B o v i n e serum albumin in comparable concentrations does not inhibit h y d r o x y l a m m o n i u m c h l o r i d e oxidation (Table 3). Nitrite reductase in the absence o f an e x o g e n o u s low potential electron donor ( N A D ( P ) H or reduced ferredoxin) has no influence on the results. Nitrate ions (tested up to 5 × 10 -4 M) do not interfere with the test system.

ACKNOWLEDGMENT We wish to thank Dr. C, Soeder for samples of deep-frozen Spirulina.

REFERENCES 1. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049. 2. Fridovich, I. (1974) in MolecuLar Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 453, Academic Press, New York. 3. Halliwell, B. (1974) New Phytol. 73, 1075. 4. Misra, H. P., and Fridovich, I. (1972) J. Biol. Chem. 247, 3170. 5. Beauchamp, C., and Fridovich, I. (1971)Anal. Biochem. 44, 276. 6. Marklund, S., and Marklund, G. (1974) Eur. J. Biochem. 47, 469. 7. Elstner, E. F., Heupel, A., and Vaklinova, S. (1970) Z. Pflanzenphysiol. 62, 184. 8. Elstner, E. F., Stoffer, C., and Heupel, A. (1975) Z. Naturforschung. 30¢, 53. 9. Elstner, E. F., and Kramer, R. (1973) Biochim. Biophys. Acta 314, 340. 10. Sawada, J., Ohyama, T., and Yamazaki, J. (1972) Biochim. Biophys. Acta 268, 305.

620 11. 12. 13. 14.

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Asada, K., Takahashi, M., and Nagate, M. (1974)Agr. Biol. Chem. 38, 471. Lumsden, J., and Hall, D. O. (1974) Biochem. Biophys. Res. Commun. 58, 35. Weisiger, R. A., and Fridovich, I. (1973)J. Biol. Chem. 248, 4793. Murphy, J. B., and Kies, M. W. (1960) Biochim. Biophys. Acta 45, 382. ERICH F. ELSTNER ADELHEID HEUPEL

Ruhr- Universiti~t Bochum Biochemie der Pflanzen P.O. Box 2148 D-4630 Bochum Received February 18, 1975; accepted September 10, 1975

Inhibition of nitrite formation from hydroxylammoniumchloride: a simple assay for superoxide dismutase.

ANALYTICAL BIOCHEMISTRY 70, 6 1 6 - - 6 2 0 (1976) Inhibition of Nitrite Formation from Hydroxylammoniumchloride: A Simple Assay for Superoxide Dismu...
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