Inflammation, Vol. 14, No. 2, 1990
METALS
INHIBIT RIBOFLAVIN-CATALYZED
GENERATION
OF SUPEROXIDE
ANION
IN
VITRO LANCE S. TERADA, JONATHAN A. LEFF, DAVID N. GUIDOT, GAYLE A. SH1BAO, and JOHN E. REPINE Department of Medicine and Webb-Waring Lung Institute University of Colorado Health Sciences Center Denver, Colorado
Abstract--We found that addition of cationic metals inhibited ravin-catalyzed superoxide anion (O1) production in vitro. Inhibition of 02 generation by metals appeared to relate to the ability of metal ions to chelate or complex with amine groups, altering their electronegativity. Metal inhibition of O~ production has important implications for biological systems involving O~- radical production, as well as for assays requiring the generation of O~- in vitro.
INTRODUCTION Superoxide anion (02) is a reactive oxygen metabolite that has been implicated in a number of basic reactions in both physiologic and disease states (2, 2). The most common biologic sources of 02 are flavin derivatives, including certain ravin-containing enzymes such as xanthine oxidase, aldehyde oxidase, and dihydroorotic dehydrogenase (3). However, 0 2 is also a product of free ravincatalyzed photooxidation of a number of amines (4, 5). This latter group of reactions uses light-activated free riboflavin to facilitate electron transfer from amine groups to 02 , resulting in amine oxidation and formation of reduced, highly reactive 02 species. This general class of reactions is thought to have been important in evolutionary chemistry and is also relevant to certain biological systems. In addition, these reactions have been used as a source of O2 for experiments in vitro and as the basis for a convenient assay for superoxide dismutase (SOD) (6). Superoxide anion is also produced by the flavin-indepen217 0360-3997/90/0400-0217506.00/0 9 1990PlenumPublishingCorporation
218
Terada et al.
dent autooxidation of amines, and this process is catalyzed by metal ions (7, 8). Hence, we sought to determine the effect of metals on the flavin-catalyzed photooxidation of amines in vitro.
M A T E R I A L S AND M E T H O D S
Reagents. Riboflavin, EDTA Na2, DL-methionine,(--)-nicotine, NaC1, phosphate salts, calcium chloride, magnesium chloride, ferric chloride, cupric sulfate, chromium chloride, manganese chloride, tungsten sodium, superoxide dismutase (SOD, bovine erythrocyte, 3000 units/ mg), and cytochrome c (horse heart, type VI) were all obtained from Sigma Chemical Co. (St. Louis, Missouri). Aluminum potassium sulfate was purchased from J.T. Baker Chemical (Phillipsburg, New Jersey) and catalase (bovineliver, 81,536 units/rag) was obtainedfrom Worthington Biochemical (Freehold, New Jersey). Generation and Measurement of 0~. O; production was measured as SOD-inhibitable reduction of cytochrome c. In a reaction volume of 1 ml, cytochrome c (1.5 mg/ml) was added to phosphate-buffered saline (20 mM, pH 7.4) containing riboflavin (1 /~M), an electron-donating amine (1 mM), and the metal salt (2 mM) in the presenceor absenceof SOD (100 #g/ml). Catalase (10/zg/ml) was added to all tubes to prevent reoxidationof cytochrome c by H202. Samples were incubated in direct sunlight for 30 min at room temperature, wrapped in foil, and placed on ice. Absorbanceat 550 nm was monitored and the rate of O~- generationcalculatedusing an extinction coefficient (reduced-oxidizedcytochrome c) of 2.10 • 104/cm/M (9). In some experiments, the concentrationsof the amine and the metal were varied from 0 to 2 mM.
RESULTS
Effect of Ca2+ on Oi Generation by Riboflavin/EDTA. The rate of production of 03- was inversely related to the concentration of added Ca 2+ ion for any given concentration of E D T A (Figure 1). Furthermore, the inhibitory effect of Ca 2§ was reversed by increasing the concentration of E D T A in the reaction mixture to a concentration greater than that of Ca z+, suggesting that inhibition may be due to chelation of Ca 2+ by E D T A . Effect of Various Metals on O~ Generation by Different Riboflavin~Amine Systems. Methionine and nicotine both caused O i to be generated at a somewhat slower rate than E D T A (Table 1). However, all of these amines were inhibited by addition of cationic metals. Addition of calcium and magnesium had a much greater inhibitory effect on E D T A than on nicotine and methionine, whereas a l u m i n u m and chromium had a lesser inhibitory effect on E D T A than on nicotine and methionine. Addition of copper and manganese strongly inhibited O i production by all three amines. By comparison, iron had a greater inhibitory effect on nicotine than on E D T A or methionine, while tungsten had only a moderate effect on both nicotine and methionine and essentially no effect
2.00 ~ 02"
1.50
(txM/min)
1.oo
9 \
•
~
~
~.~,1000gM
7~o
~ooo
~ 7 5 0 gM
0.50
0
o
2~;o
s~o
[Ca++] ( ~ ) Fig. 1. Effect of Ca 2+ on the rate of O~ production by riboflavin-catalyzed EDTA photooxidation. O~- generation decreases with increasing Ca 2+ concentrations. This effect is obviated by increasing EDTA concentration. Values are means of four individual determinations. Table 1. Effect of Metal Ions on O~- Generation by Photooxidation of EDTA, Nicotine, and Methionine" EDTA
Nicotine
Methionine
Control
2.00 _+ 0.05 (100%)
0.84 4- 0.09 (100%)
1.00 _+ 0.02 (100%)
Ca 2+
0.26 4- 0.03* (13%)
0.50 • 0.04* (60%)
0.81 + 0.08* (81%)
Mg 2§
0.22 + 0.03* (11%)
0.50 + 0.04* (60%)
0.68 4- 0.02* (68%)
Cr 2+
1.79 • 0.06* (90%)
0.22 4- 0.03* (26%)
0.40 + 0.04* (40%)
A1z+
1.44 + 0.13" (72%)
0.14 4- 0.05* (17%)
0.29 + 0.02* (29%)
Cu 3+
0.02 + 0.01" (1%)
0.02 4- 0.02* (2%)
0 4- 0* (0%)
Mn 2+
0.01 • 0.01" (0.5%)
0.04 4- 0.04 (5%)
0 • 0 (0%)
Fe 3+
1.08 4- 0.05* (54%)
0.02 4- 0.02* (2%)
0.50 + 0.04* (50%)
W 6+
1.85 • 0.01 (93%)
0.52 + 0.08 (62%)
0.37 • 0.02* (37%)
~Rate of 03- generation (/zM/min) from riboflavin (1 #M), amine (1 mM), and sunlight, in the presence or absence of various metals (2 raM). Values are the mean + SE of four individual determinations. Values in parentheses are percent of control for each amine tested. *p < 0.05 compared with control for each amine.
220
Teradaetal.
on EDTA. Inhibition of cytochrome c reduction was not due to direct oxidation of cytochrome c following addition of metal salts (data not shown).
DISCUSSION We found that addition of cationic metal ions inhibited production of Of by riboflavin-catalyzed photooxidation of amines. Studies of the stoichiometry of the interaction between EDTA and Ca 2+ suggested that the inhibitory effect may be due to chelation of the metal with the amine. Although soluble metals generally possess prooxidant properties by virtue of their ability to facilitate electron transfer, in this case metals inhibited the formation of 0 2 . Thus, it appears that trace levels of metals can promote autooxidation of certain amines and thiols (7, 8), while higher concentrations can inhibit their riboflavin-catalyzed photooxidation. Since the susceptibility of a nitrogen compound to photooxidation by riboflavin and light increases as the electronegativity of the amine group increases (quaternary < primary < secondary < tertiary amine) (5), it is not surprising that complexing the amine group with a cationic metal would greatly decrease the availability of the electrons at the nitrogen atom. Accordingly, the extent of interference with the generation of O f may be an indicator of the chelating strength of the amine group for the metal. Hence, Ca 2+ and Mg 2+ strongly interact with EDTA and greatly decrease its photoxidation, whereas methionine and nicotine appear to be weaker chelators and O f production more weakly inhibited. Photooxidation reactions appear to be important in biological systems. Amine photooxidation, for instance, has been implicated in evolutionary chemistry. Formaldehyde, a product of photoxidation, is regarded as important in the origin of life, and certain amines involved as reactants are thought to have been early organic compounds present in the earth's primordial atmosphere (5). In addition, a similar phenomenon occurs with the plant flavoprotein indole acetic acid oxidase. Since indole acetic acid is a growth horomone, this lightactivated, riboflavin-catalyzed reaction has been implicated in phototropism (5). Finally, the mechanism by which pipe-smoking causes oral cancer remains obscure, but might conceivably involve radical generation by flavins and nicotine. All of these postulated reactions could be profoundly affected and perhaps even modulated by the local concentration of soluble metals. The reported phenomenon has practical implications insofar as this general reaction is commonly used as a source of O f in an assay of SOD (6). This reaction is also used to assess the susceptibility of certain targets to O~- (10).
Metals Inhibit Superoxide Generation
221
A s a r e s u l t , t h e u n r e c o g n i z e d p r e s e n c e o f m e t a l s , s u c h as C a 2+ o r M g 2+, in a buffer or tissue sample may falsely elevate measurements of SOD activity and/ o r d e c r e a s e a t a r g e t m o l e c u l e ' s a p p a r e n t s u s c e p t i b i l i t y to O ~ . Acknowledgments--This work was supported in part by grants from the National Institutes of Health (PO1-AM 35098, RO1-HL 35378, RO1-HL 28182, PO1-HL 27353, and RO1-HL 34486), American Heart Association, American Lung Association, and the Swan Foundation.
REFERENCES
1. McCoRD, J. M. 1985. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159. 2. FREEMAN,B. A., and J. D. CRAPO. 1982. Free radicals and tissue injury. Lab, Invest. 47:412. 3. RAJAGOPALAN,K. V., and P. HANDLER, 1968. Metalloflavoproteins. In Biological Oxidations. T.P. Singer, editor. Interscience Publishers, New York. 301. 4. MASSEY,V., S. STRICKLAND,S. G. MAYHEW,L. G. HOWELL, P. C. ENGEL, R. G. MATTHEWS, M. SCHUMAN, and P. A. SULLIVAN. 1969. The production of superoxide anion radicals in the reaction of reduced flavins and flavoproteins with molecular oxygen. Biochem. Biophys. Res. Commun. 36:891. 5. FRISELL,W. R., C. W. CHUNG,and C. G. MACKENZIE. 1959. Catalysis of oxidation of nitrogen compounds by ravin coenzymes in the presence of light. J. Biol. Chem. 234:1297. 6. BEAUCHAMP, C., and I. FRIDOVICH. 1971. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276. 7. MISRA, H. P., and I. FRIDOVICH. 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247:3170. 8. MISRA, H. P. 1974. Generation of superoxide free radical during the autoxidation of thiols. J. Biol. Chem. 249:2151. 9. MASSEY, V. 1959. The microestimation of succinate and the extinction coefficiem of cytochrome c. Biochim. Biophys. Acta 34:255. 10. TERADA,L. S., C. J. BEEHLER,A. BANERJEE,J. M. BROWN,M. A. GROSSO, A. H. HARKEN', J. M. McCoRD, and J. E. REPINE. 1988. Hyperoxia and self- or neutrophil-generated 02 metabolites inactivate xanthine oxidase. J. Appl. Physiol. 65:2349.
METALS
INHIBIT RIBOFLAVIN-CATALYZED
GENERATION
OF SUPEROXIDE
ANION
IN
VITRO LANCE S. TERADA, JONATHAN A. LEFF, DAVID N. GUIDOT, GAYLE A. SH1BAO, and JOHN E. REPINE Department of Medicine and Webb-Waring Lung Institute University of Colorado Health Sciences Center Denver, Colorado
Abstract--We found that addition of cationic metals inhibited ravin-catalyzed superoxide anion (O1) production in vitro. Inhibition of 02 generation by metals appeared to relate to the ability of metal ions to chelate or complex with amine groups, altering their electronegativity. Metal inhibition of O~ production has important implications for biological systems involving O~- radical production, as well as for assays requiring the generation of O~- in vitro.
INTRODUCTION Superoxide anion (02) is a reactive oxygen metabolite that has been implicated in a number of basic reactions in both physiologic and disease states (2, 2). The most common biologic sources of 02 are flavin derivatives, including certain ravin-containing enzymes such as xanthine oxidase, aldehyde oxidase, and dihydroorotic dehydrogenase (3). However, 0 2 is also a product of free ravincatalyzed photooxidation of a number of amines (4, 5). This latter group of reactions uses light-activated free riboflavin to facilitate electron transfer from amine groups to 02 , resulting in amine oxidation and formation of reduced, highly reactive 02 species. This general class of reactions is thought to have been important in evolutionary chemistry and is also relevant to certain biological systems. In addition, these reactions have been used as a source of O2 for experiments in vitro and as the basis for a convenient assay for superoxide dismutase (SOD) (6). Superoxide anion is also produced by the flavin-indepen217 0360-3997/90/0400-0217506.00/0 9 1990PlenumPublishingCorporation
218
Terada et al.
dent autooxidation of amines, and this process is catalyzed by metal ions (7, 8). Hence, we sought to determine the effect of metals on the flavin-catalyzed photooxidation of amines in vitro.
M A T E R I A L S AND M E T H O D S
Reagents. Riboflavin, EDTA Na2, DL-methionine,(--)-nicotine, NaC1, phosphate salts, calcium chloride, magnesium chloride, ferric chloride, cupric sulfate, chromium chloride, manganese chloride, tungsten sodium, superoxide dismutase (SOD, bovine erythrocyte, 3000 units/ mg), and cytochrome c (horse heart, type VI) were all obtained from Sigma Chemical Co. (St. Louis, Missouri). Aluminum potassium sulfate was purchased from J.T. Baker Chemical (Phillipsburg, New Jersey) and catalase (bovineliver, 81,536 units/rag) was obtainedfrom Worthington Biochemical (Freehold, New Jersey). Generation and Measurement of 0~. O; production was measured as SOD-inhibitable reduction of cytochrome c. In a reaction volume of 1 ml, cytochrome c (1.5 mg/ml) was added to phosphate-buffered saline (20 mM, pH 7.4) containing riboflavin (1 /~M), an electron-donating amine (1 mM), and the metal salt (2 mM) in the presenceor absenceof SOD (100 #g/ml). Catalase (10/zg/ml) was added to all tubes to prevent reoxidationof cytochrome c by H202. Samples were incubated in direct sunlight for 30 min at room temperature, wrapped in foil, and placed on ice. Absorbanceat 550 nm was monitored and the rate of O~- generationcalculatedusing an extinction coefficient (reduced-oxidizedcytochrome c) of 2.10 • 104/cm/M (9). In some experiments, the concentrationsof the amine and the metal were varied from 0 to 2 mM.
RESULTS
Effect of Ca2+ on Oi Generation by Riboflavin/EDTA. The rate of production of 03- was inversely related to the concentration of added Ca 2+ ion for any given concentration of E D T A (Figure 1). Furthermore, the inhibitory effect of Ca 2§ was reversed by increasing the concentration of E D T A in the reaction mixture to a concentration greater than that of Ca z+, suggesting that inhibition may be due to chelation of Ca 2+ by E D T A . Effect of Various Metals on O~ Generation by Different Riboflavin~Amine Systems. Methionine and nicotine both caused O i to be generated at a somewhat slower rate than E D T A (Table 1). However, all of these amines were inhibited by addition of cationic metals. Addition of calcium and magnesium had a much greater inhibitory effect on E D T A than on nicotine and methionine, whereas a l u m i n u m and chromium had a lesser inhibitory effect on E D T A than on nicotine and methionine. Addition of copper and manganese strongly inhibited O i production by all three amines. By comparison, iron had a greater inhibitory effect on nicotine than on E D T A or methionine, while tungsten had only a moderate effect on both nicotine and methionine and essentially no effect
2.00 ~ 02"
1.50
(txM/min)
1.oo
9 \
•
~
~
~.~,1000gM
7~o
~ooo
~ 7 5 0 gM
0.50
0
o
2~;o
s~o
[Ca++] ( ~ ) Fig. 1. Effect of Ca 2+ on the rate of O~ production by riboflavin-catalyzed EDTA photooxidation. O~- generation decreases with increasing Ca 2+ concentrations. This effect is obviated by increasing EDTA concentration. Values are means of four individual determinations. Table 1. Effect of Metal Ions on O~- Generation by Photooxidation of EDTA, Nicotine, and Methionine" EDTA
Nicotine
Methionine
Control
2.00 _+ 0.05 (100%)
0.84 4- 0.09 (100%)
1.00 _+ 0.02 (100%)
Ca 2+
0.26 4- 0.03* (13%)
0.50 • 0.04* (60%)
0.81 + 0.08* (81%)
Mg 2§
0.22 + 0.03* (11%)
0.50 + 0.04* (60%)
0.68 4- 0.02* (68%)
Cr 2+
1.79 • 0.06* (90%)
0.22 4- 0.03* (26%)
0.40 + 0.04* (40%)
A1z+
1.44 + 0.13" (72%)
0.14 4- 0.05* (17%)
0.29 + 0.02* (29%)
Cu 3+
0.02 + 0.01" (1%)
0.02 4- 0.02* (2%)
0 4- 0* (0%)
Mn 2+
0.01 • 0.01" (0.5%)
0.04 4- 0.04 (5%)
0 • 0 (0%)
Fe 3+
1.08 4- 0.05* (54%)
0.02 4- 0.02* (2%)
0.50 + 0.04* (50%)
W 6+
1.85 • 0.01 (93%)
0.52 + 0.08 (62%)
0.37 • 0.02* (37%)
~Rate of 03- generation (/zM/min) from riboflavin (1 #M), amine (1 mM), and sunlight, in the presence or absence of various metals (2 raM). Values are the mean + SE of four individual determinations. Values in parentheses are percent of control for each amine tested. *p < 0.05 compared with control for each amine.
220
Teradaetal.
on EDTA. Inhibition of cytochrome c reduction was not due to direct oxidation of cytochrome c following addition of metal salts (data not shown).
DISCUSSION We found that addition of cationic metal ions inhibited production of Of by riboflavin-catalyzed photooxidation of amines. Studies of the stoichiometry of the interaction between EDTA and Ca 2+ suggested that the inhibitory effect may be due to chelation of the metal with the amine. Although soluble metals generally possess prooxidant properties by virtue of their ability to facilitate electron transfer, in this case metals inhibited the formation of 0 2 . Thus, it appears that trace levels of metals can promote autooxidation of certain amines and thiols (7, 8), while higher concentrations can inhibit their riboflavin-catalyzed photooxidation. Since the susceptibility of a nitrogen compound to photooxidation by riboflavin and light increases as the electronegativity of the amine group increases (quaternary < primary < secondary < tertiary amine) (5), it is not surprising that complexing the amine group with a cationic metal would greatly decrease the availability of the electrons at the nitrogen atom. Accordingly, the extent of interference with the generation of O f may be an indicator of the chelating strength of the amine group for the metal. Hence, Ca 2+ and Mg 2+ strongly interact with EDTA and greatly decrease its photoxidation, whereas methionine and nicotine appear to be weaker chelators and O f production more weakly inhibited. Photooxidation reactions appear to be important in biological systems. Amine photooxidation, for instance, has been implicated in evolutionary chemistry. Formaldehyde, a product of photoxidation, is regarded as important in the origin of life, and certain amines involved as reactants are thought to have been early organic compounds present in the earth's primordial atmosphere (5). In addition, a similar phenomenon occurs with the plant flavoprotein indole acetic acid oxidase. Since indole acetic acid is a growth horomone, this lightactivated, riboflavin-catalyzed reaction has been implicated in phototropism (5). Finally, the mechanism by which pipe-smoking causes oral cancer remains obscure, but might conceivably involve radical generation by flavins and nicotine. All of these postulated reactions could be profoundly affected and perhaps even modulated by the local concentration of soluble metals. The reported phenomenon has practical implications insofar as this general reaction is commonly used as a source of O f in an assay of SOD (6). This reaction is also used to assess the susceptibility of certain targets to O~- (10).
Metals Inhibit Superoxide Generation
221
A s a r e s u l t , t h e u n r e c o g n i z e d p r e s e n c e o f m e t a l s , s u c h as C a 2+ o r M g 2+, in a buffer or tissue sample may falsely elevate measurements of SOD activity and/ o r d e c r e a s e a t a r g e t m o l e c u l e ' s a p p a r e n t s u s c e p t i b i l i t y to O ~ . Acknowledgments--This work was supported in part by grants from the National Institutes of Health (PO1-AM 35098, RO1-HL 35378, RO1-HL 28182, PO1-HL 27353, and RO1-HL 34486), American Heart Association, American Lung Association, and the Swan Foundation.
REFERENCES
1. McCoRD, J. M. 1985. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159. 2. FREEMAN,B. A., and J. D. CRAPO. 1982. Free radicals and tissue injury. Lab, Invest. 47:412. 3. RAJAGOPALAN,K. V., and P. HANDLER, 1968. Metalloflavoproteins. In Biological Oxidations. T.P. Singer, editor. Interscience Publishers, New York. 301. 4. MASSEY,V., S. STRICKLAND,S. G. MAYHEW,L. G. HOWELL, P. C. ENGEL, R. G. MATTHEWS, M. SCHUMAN, and P. A. SULLIVAN. 1969. The production of superoxide anion radicals in the reaction of reduced flavins and flavoproteins with molecular oxygen. Biochem. Biophys. Res. Commun. 36:891. 5. FRISELL,W. R., C. W. CHUNG,and C. G. MACKENZIE. 1959. Catalysis of oxidation of nitrogen compounds by ravin coenzymes in the presence of light. J. Biol. Chem. 234:1297. 6. BEAUCHAMP, C., and I. FRIDOVICH. 1971. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276. 7. MISRA, H. P., and I. FRIDOVICH. 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247:3170. 8. MISRA, H. P. 1974. Generation of superoxide free radical during the autoxidation of thiols. J. Biol. Chem. 249:2151. 9. MASSEY, V. 1959. The microestimation of succinate and the extinction coefficiem of cytochrome c. Biochim. Biophys. Acta 34:255. 10. TERADA,L. S., C. J. BEEHLER,A. BANERJEE,J. M. BROWN,M. A. GROSSO, A. H. HARKEN', J. M. McCoRD, and J. E. REPINE. 1988. Hyperoxia and self- or neutrophil-generated 02 metabolites inactivate xanthine oxidase. J. Appl. Physiol. 65:2349.
