220
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
[19]
References Ranges
The specific activities of total SOD from various sources, according to the present method, are tabulated below. These values are intended only to provide references for practical purposes, and a certain degree of variability may be expected depending on the material and treatments employed. Source
Activity
Human plasma Human erythrocytes Human erythrocytes Human mononuclear leukocytes Rat liver (perfused) HL-60 (human promyelocytic cells) 3T3-BALB (normal fibroblasts) 3T3-B77 (transformed fibroblasts) NIH-3T3 KiMSV (transformed fibroblasts) Friend erythroleukemia cells Cu,Zn-SOD (Boehringer; Cat. 837 113)
0.33 U/rag protein 11.5 U/mg Hb 0.36 U/106 cells 20 U/rag cytosolic protein 324 U/mg cytosolic protein 37 U/mg cytosolic protein 63 U/mg cytosolic protein 27 U/mg cytosolic protein 26 U/mg cytosolic protein 45 U/rag cytosolic protein 32,180 U/rag lyophilizate
Acknowledgements This work was supported by grants from the Ministero della Pubblica Istruzione (60 and 40%) and from the Associazione Italianaper la Riccrca sul Cancro (A.I.R.C.).
[19] A s s a y s 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 on Autoxidation of H e m a t o x y l i n By JOSEPH P. MARTIN, JR.
Introduction Superoxide dismutase
(SOD) catalyzes the r e a c t i o n
02- + 02- + 2 H + --~ H20~ + 02
(1)
A variety of spectrophotometric assays for the enzyme have been described. O n e c a t e g o r y o f a s s a y s c o m b i n e s superoxide-generating sysMETHODS 1N ENZYMOLOGY, VOL. 186
Copyright © 1990by AcademicPress, Inc. All fights of reproductionin any form reserved.
[19]
POSITIVE AND NEGATIVE S O D ASSAYS
221
tems, such as xanthine oxidase, ~ alkaline dimethyl sulfoxide, 2 and photochemical oxidation of reduced substrates, 3 with an indicating scavenger of superoxide. These scavengers include cytochrome c, 1 hydroxylamine, 4 and nitro blue tetrazolium) In these assays superoxide dismutase activity is quantitated by determining the ability of the enzyme to compete with the scavenger for the available 02-. In contrast, autoxidation assays can be performed in which the oxidizing species is both a source of O2- and an indicating scavenger for 02-. Assays based on the autoxidation of pyrogallol, 5,6 hydroxylamine, 7 6-hydroxydopamine, 8 and epinephrine 9,1° are simple to perform, involve few reaction components, and are easily monitored in the visible region of the absorption spectrum. One disadvantage of autoxidation assays is that most must be carried out at high pH where the specific activity of manganese and iron SODs is low. H In this chapter, I describe a new autoxidation assay based on the transformation of hematoxylin to its two-electron-oxidized product, hematein. The assay is sensitive and simple. The reaction product, hemarein, is relatively stable and has an absorption maximum and extinction coefficient similar to those of the commonly used 02- scavenger, cytochrome c. The reaction is inhibited by SOD and can be performed in the physiological pH range of 6.8-7.8 where SOD inhibits the reaction 9095%. In addition, at pH values above 8.1, the autoxidation reaction is accelerated by added SOD, and the reaction becomes a positive assay for the enzyme, similar in principle to SOD assays based on the photochemical and enzymatic oxidation of dianisidine. 12,~3 Materials and Methods
The activity of purified SOD is determined by the xanthine oxidasecytochrome c method.1 Xanthine oxidase is isolated from unpasteurized 1 j. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 2 K. Hyland, E. Voisin, H. Banoun, and C. Auclair, Anal. Biochem. 135, 280 (1983). 3 C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971). 4 E. Elstner and A. Heupel, Anal. Biochem. 70, 616 (1976). S. Marklund and G. Marklund, Eur. J. Biochem. 47, 469 (1974). 6 S. Marklund, in "CRC Handbook of Oxygen Radical Research" (R. Greenwald, ed.), p. 243. CRC Press, Boca Raton, Florida, 1985. 7 y . Kono, Arch. Biochem. Biophys. 186, 189 (1978). s R. E. Heikkela and F. S. Cabbat, Anal. Biochem. 75, 356 (1976). 9 H. P. Misra and I. Fridovich, J. Biol. Chem. 247, 3170 (1972). l0 M. Sun and S. Zigman, Anal. Biochem. 90, 81 (1981). " H. J. Forman and I. Fridovich, Arch. Biochem. Biophys. 159, 396 (1973). 12 H. P. Misra and I. Fridovich, Arch. Biochem. Biophys. 181, 308 (1977). 13 H. P. Misra and I. Fridovich, Anal. Biochem. 79, 553 (1977).
222
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
[19]
cream. TM General protein is determined by the Bradford assay using bovine serum albumin (BSA) as a standard. ~5 Hematoxylin Assay. Hematoxylin is made up as a 1-5 mM stock solution in 50 mM monobasic potassium phosphate buffer. Reactions are started by adding aliquots of this stock solution to 3 ml of 50 mM potassium phosphate, 0.1 mM ethylenediaminetetraacetic acid (EDTA), at the indicated pH at 25 °. Autoxidation is monitored as an increase in the absorbance at 560 nm in an IBM Model 9420 UV-visible spectrophotometer. The rates reported represent those obtained over the first 4 rain of reaction. Anaerobic measurements are made in anaerobic quartz cuvettes. ~6 The buffer is scrubbed for 25 min with ultrapure nitrogen, and hematoxylin is tipped into the buffer from a sidearm. SOD inhibition studies are carried out by adding aliquots of purified copper zinc superoxide dismutase (Cu,Zn-SOD) solution to the hematoxylin autoxidation assay. The SOD solution is precalibrated using the xanthine oxidase-cytochrome c method.l Results
Hematoxylin Autoxidation. When hematoxylin dissolved in an acidic stock solution is diluted into 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.5, 25°), its autoxidation is detectable as an increase in absorbance between 400 and 670 nm (Fig. 1). The spectra of the oxidized substrate are identical to those of the oxidation product, hematein, and show an absorption maximum at 558 nm. The anaerobic spectrum, measured after 3 hr of incubation, is similar to the aerobic spectrum obtained immediately following dilution. Hematein in 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.5) has an A560 extinction coefficient of 27,000 M -~ cm -1. The autoxidation rate, estimated at 560 nm, is first order with respect to hematoxylin concentration and is pH dependent. At pH 6.5 the autoxidation of 66/xM hematoxylin is nearly undetectable, but the rate steadily increases with increasing pH and at pH 8.9 is 0.293 As~min. Hematoxylin autoxidation at the low end of the pH range is easily observed by increasing the hematoxylin concentration in the assay. For example, at pH 7.2 a rate of 0.022 A560/min is obtained at a hematoxylin concentration of 165 /xM, whereas 72/zM gives the same reaction rate at pH 7.5. Effects ofSuperoxide Dismutase. SOD inhibits hematoxylin autoxida14 W. A. Waud, F. D. Brady, R. D. Wiley, and K. V. Rajagopalan, Arch. Biochem. Biophys. 169, 695 (1975). 15 M. Bradford, Anal. Biochem. 72, 248 (1976). 16 E. K. Hodgson, J. M. McCord, and I. Fridovich, Anal. Biochem. 5, 470 (1973).
[19]
POSITIVE AND NEGATIVE S O D ASSAYS
223
0.8 0.7 120 rain
0.6
0.5 o ¢-
0 (/) .,¢3
0.4
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
[19]
References Ranges
The specific activities of total SOD from various sources, according to the present method, are tabulated below. These values are intended only to provide references for practical purposes, and a certain degree of variability may be expected depending on the material and treatments employed. Source
Activity
Human plasma Human erythrocytes Human erythrocytes Human mononuclear leukocytes Rat liver (perfused) HL-60 (human promyelocytic cells) 3T3-BALB (normal fibroblasts) 3T3-B77 (transformed fibroblasts) NIH-3T3 KiMSV (transformed fibroblasts) Friend erythroleukemia cells Cu,Zn-SOD (Boehringer; Cat. 837 113)
0.33 U/rag protein 11.5 U/mg Hb 0.36 U/106 cells 20 U/rag cytosolic protein 324 U/mg cytosolic protein 37 U/mg cytosolic protein 63 U/mg cytosolic protein 27 U/mg cytosolic protein 26 U/mg cytosolic protein 45 U/rag cytosolic protein 32,180 U/rag lyophilizate
Acknowledgements This work was supported by grants from the Ministero della Pubblica Istruzione (60 and 40%) and from the Associazione Italianaper la Riccrca sul Cancro (A.I.R.C.).
[19] A s s a y s 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 on Autoxidation of H e m a t o x y l i n By JOSEPH P. MARTIN, JR.
Introduction Superoxide dismutase
(SOD) catalyzes the r e a c t i o n
02- + 02- + 2 H + --~ H20~ + 02
(1)
A variety of spectrophotometric assays for the enzyme have been described. O n e c a t e g o r y o f a s s a y s c o m b i n e s superoxide-generating sysMETHODS 1N ENZYMOLOGY, VOL. 186
Copyright © 1990by AcademicPress, Inc. All fights of reproductionin any form reserved.
[19]
POSITIVE AND NEGATIVE S O D ASSAYS
221
tems, such as xanthine oxidase, ~ alkaline dimethyl sulfoxide, 2 and photochemical oxidation of reduced substrates, 3 with an indicating scavenger of superoxide. These scavengers include cytochrome c, 1 hydroxylamine, 4 and nitro blue tetrazolium) In these assays superoxide dismutase activity is quantitated by determining the ability of the enzyme to compete with the scavenger for the available 02-. In contrast, autoxidation assays can be performed in which the oxidizing species is both a source of O2- and an indicating scavenger for 02-. Assays based on the autoxidation of pyrogallol, 5,6 hydroxylamine, 7 6-hydroxydopamine, 8 and epinephrine 9,1° are simple to perform, involve few reaction components, and are easily monitored in the visible region of the absorption spectrum. One disadvantage of autoxidation assays is that most must be carried out at high pH where the specific activity of manganese and iron SODs is low. H In this chapter, I describe a new autoxidation assay based on the transformation of hematoxylin to its two-electron-oxidized product, hematein. The assay is sensitive and simple. The reaction product, hemarein, is relatively stable and has an absorption maximum and extinction coefficient similar to those of the commonly used 02- scavenger, cytochrome c. The reaction is inhibited by SOD and can be performed in the physiological pH range of 6.8-7.8 where SOD inhibits the reaction 9095%. In addition, at pH values above 8.1, the autoxidation reaction is accelerated by added SOD, and the reaction becomes a positive assay for the enzyme, similar in principle to SOD assays based on the photochemical and enzymatic oxidation of dianisidine. 12,~3 Materials and Methods
The activity of purified SOD is determined by the xanthine oxidasecytochrome c method.1 Xanthine oxidase is isolated from unpasteurized 1 j. M. McCord and I. Fridovich, J. Biol. Chem. 244, 6049 (1969). 2 K. Hyland, E. Voisin, H. Banoun, and C. Auclair, Anal. Biochem. 135, 280 (1983). 3 C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971). 4 E. Elstner and A. Heupel, Anal. Biochem. 70, 616 (1976). S. Marklund and G. Marklund, Eur. J. Biochem. 47, 469 (1974). 6 S. Marklund, in "CRC Handbook of Oxygen Radical Research" (R. Greenwald, ed.), p. 243. CRC Press, Boca Raton, Florida, 1985. 7 y . Kono, Arch. Biochem. Biophys. 186, 189 (1978). s R. E. Heikkela and F. S. Cabbat, Anal. Biochem. 75, 356 (1976). 9 H. P. Misra and I. Fridovich, J. Biol. Chem. 247, 3170 (1972). l0 M. Sun and S. Zigman, Anal. Biochem. 90, 81 (1981). " H. J. Forman and I. Fridovich, Arch. Biochem. Biophys. 159, 396 (1973). 12 H. P. Misra and I. Fridovich, Arch. Biochem. Biophys. 181, 308 (1977). 13 H. P. Misra and I. Fridovich, Anal. Biochem. 79, 553 (1977).
222
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
[19]
cream. TM General protein is determined by the Bradford assay using bovine serum albumin (BSA) as a standard. ~5 Hematoxylin Assay. Hematoxylin is made up as a 1-5 mM stock solution in 50 mM monobasic potassium phosphate buffer. Reactions are started by adding aliquots of this stock solution to 3 ml of 50 mM potassium phosphate, 0.1 mM ethylenediaminetetraacetic acid (EDTA), at the indicated pH at 25 °. Autoxidation is monitored as an increase in the absorbance at 560 nm in an IBM Model 9420 UV-visible spectrophotometer. The rates reported represent those obtained over the first 4 rain of reaction. Anaerobic measurements are made in anaerobic quartz cuvettes. ~6 The buffer is scrubbed for 25 min with ultrapure nitrogen, and hematoxylin is tipped into the buffer from a sidearm. SOD inhibition studies are carried out by adding aliquots of purified copper zinc superoxide dismutase (Cu,Zn-SOD) solution to the hematoxylin autoxidation assay. The SOD solution is precalibrated using the xanthine oxidase-cytochrome c method.l Results
Hematoxylin Autoxidation. When hematoxylin dissolved in an acidic stock solution is diluted into 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.5, 25°), its autoxidation is detectable as an increase in absorbance between 400 and 670 nm (Fig. 1). The spectra of the oxidized substrate are identical to those of the oxidation product, hematein, and show an absorption maximum at 558 nm. The anaerobic spectrum, measured after 3 hr of incubation, is similar to the aerobic spectrum obtained immediately following dilution. Hematein in 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.5) has an A560 extinction coefficient of 27,000 M -~ cm -1. The autoxidation rate, estimated at 560 nm, is first order with respect to hematoxylin concentration and is pH dependent. At pH 6.5 the autoxidation of 66/xM hematoxylin is nearly undetectable, but the rate steadily increases with increasing pH and at pH 8.9 is 0.293 As~min. Hematoxylin autoxidation at the low end of the pH range is easily observed by increasing the hematoxylin concentration in the assay. For example, at pH 7.2 a rate of 0.022 A560/min is obtained at a hematoxylin concentration of 165 /xM, whereas 72/zM gives the same reaction rate at pH 7.5. Effects ofSuperoxide Dismutase. SOD inhibits hematoxylin autoxida14 W. A. Waud, F. D. Brady, R. D. Wiley, and K. V. Rajagopalan, Arch. Biochem. Biophys. 169, 695 (1975). 15 M. Bradford, Anal. Biochem. 72, 248 (1976). 16 E. K. Hodgson, J. M. McCord, and I. Fridovich, Anal. Biochem. 5, 470 (1973).
[19]
POSITIVE AND NEGATIVE S O D ASSAYS
223
0.8 0.7 120 rain
0.6
0.5 o ¢-
0 (/) .,¢3
0.4
