(.1 ./ (9.," A; ) “I ‘ ABSTRACT SUPEROXIDE DISMUTASES AND SUPEROXIDE RADICAL: OCCURRENCE IN HIGHER PLANTS AND POSSIBLE ROLE IN THE ACTION OF HERBICIDES By Constantine Nicholas Giannopolitis The photochemical assay for superoxide dismutase (SOD) consisting of methionine, riboflavin, and p-nitro blue tetrazolium chloride (NBT) was adjusted for quantitation of the enzyme in crude extracts. The enzyme units can be accurately determined from the ratio of NBT reduction in absence versus in presence of SOD and not from the percent inhibition of NBT reduction. An equation derived from the kinetics of the reaction and confirmed with various crude extracts can be used for calculating enzyme units from the above ratio. Interferences with enzyme assays of crude extracts were examined. Only peroxidase at high concentrations was shown to interfere. Peroxidase was easily inactivated by heat and SOD was heat- stable, allowing correction for peroxidase interference by heating the crude extracts. Shoots, roots, and seeds of corn (Zea mays L., cv. Michigan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were analyzed for their SOD content. The enzyme is present in the shoots, roots and seeds of all three species. Quantitative differ- ences exist between species and between organs within a species. On a dry weight basis, shoots contain more enzyme than roots. In seeds, Constantine Nicholas Giannopolitis the enzyme is present in both the embryo and the storage tissue. It was estimated that SOD accounts for 0.9 to 3.1% of the water-soluble protein in lO-day-old seedlings of corn, oats, and peas. The specific activity of SOD increased 3-fold during germination of oats, and 40% during green- ing and hook opening of the pea plumule. Electrophoresis indicated multiple forms of the enzyme. Ten distinct enzyme bands were obtained from the three species. Corn contained seven of the bands and oats three different bands. Peas contained one of the corn, and two of the oat enzymes. Some of the SOD forms were found pri- marily in mitochondria or chloroplasts. Differences and similarities in the enzyme pattern of the various organs may be explained by the organelle specificity of the SOD forms. Superoxide dismutase was purified to a maximum specific activity from pea seeds, and partially purified from corn seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct SOD bands char- acterizing the crude pea extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are isozymes of a cupro- zinc SOD. One of the remaining corn bands was shown to be a peroxidase. The other was a protein resistant to cyanide and sensitive to chloroform- ethanol treatments and may be a manganese-containing SOD. The ability of herbicides to produce superoxide radical as well as their ability to react with this radical was examined through their effect on the superoxide-induced reduction of NBT. Paraquat enhanced and diuron inhibited the reduction of NBT. Paraquat was reduced photochemically Constantine Nicholas Giannopolitis (riboflavin/methionine) or enzymatically (xanthine/xanthine oxidase) and produced superoxide radical upon reoxidation. Diuron and monuron inter- acted with photochemically produced superoxide radical, but not with enzymatically produced superoxide radical. The product of the monuron/ superoxide interaction was a demethylated, dechlorinated water-soluble compound containing phenolic hydroxyl group(s), which was not toxic to oats. The enzyme SOD prevented the formation of this product. Other herbicides (atrazine, metribuzin, terbacil, 2,4-D, CDEC, diphenamid) had little effect on the NBT reduction. SUPEROXIDE DISMUTASE AND SUPEROXIDE RADICAL: OCCURRENCE IN HIGHER PLANTS AND POSSIBLE ROLE IN THE ACTION OF HERBICIDES By Constantine Nicholas Giannopolitis A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture I976 ACKNOWLEDGEMENTS The author wishes to express his sincere thanks to Dr. S.K. Ries for his guidance and encouragement throughout this study. Appreciation is also expressed to Drs. N.E. Good, D. Penner, M.J. Zabik, G.S. Ayers, and H.J. Carew for serving on the guidance committee and for advice. Appreciation is further due to Mrs. V. Wert and Dr. G.S. Ayers, for technical assistance, and to Dr. R.A. Leavitt for assistance with the OLE and mass-spectrometric analyses. Mrs. J. Fortman's patience in typing this thesis is also appreciated. ii TABLE OF CONTENTS Page LIST OF TABLES LIST OF FIGURES INTRODUCTION ............................ l LITERATURE REVIEW .......................... 3 THE SUPEROXIDE RADICAL (02") ................. 3 Forms of Oxygen. _ .................... 3 Generation of 02'_ .................... 4 Properties of 02' ...... _ .............. 4 Biological Significance of 02° .............. 5 SUPEROXIDE DISMUTASES (SOD) .................. 6 Occurrence ........................ 6 Typesand Isozymes ..................... 7 Induction ......................... 9 Biological Role ...................... lO SUPEROXIDE RADICAL AND HERBICIDES .............. ll LITERATURE CITED ........................ l4 SECTION ONE: OCCURRENCE OF SUPEROXIDE DISMUTASES IN HIGHER PLANTS . 21 ABSTRACT ............................ 22 INTRODUCTION .......................... 23 MATERIALS AND METHODS ..................... 24 Plant Material ...................... 24 Preparation of Extracts .................. 24 Isolation of Organelles .................. 26 Electrophoresis ...................... 26 Enzyme Assay ....................... 26 RESULTS AND DISCUSSION .................... Enzyme Quantitation ................... Superoxide Dismutase Content of Plant Organs ....... and Tissues ...................... Multiple Forms of SOD ................... LITERATURE CITED ....................... SECTION TWO: PURIFICATION AND PROPERTIES OF SUPEROXIDE DISMUTASE FROM HIGHER PLANTS ......................... ABSTRACT ........................... INTRODUCTION ......................... MATERIALS AND METHODS ..................... Enzyme Purification ................... Interference Experiments ................. Changes of SOD Specific Activity ............. RESULTS AND DISCUSSION .................... Enzyme Purification ................... Seedling Enzyme as Percentage of the Water-soluble Protein ........................ Interferences with Enzyme Activity in Crude Extract Assays .................... Changes in Specific Activity of SOD ........... LITERATURE CITED ....................... SECTION THREE: IN VITRO PRODUCTIONS OF SUPEROXIDE RADICAL FROM PARAQUAT AND ITS INTERACTION WITH MONURON AND DIURON ....... ABSTRACT ........................... INTRODUCTION ......................... MATERIALS AND METHODS .................... RESULTS AND DISCUSSION .................... Effect of Herbicides on the Reduction of NBT ...... Production of 02° from Paraquat» . . . . ; ....... Interaction of Diuron and Monuron with 02' ....... LITERATURE CITED ....................... SUMMARY AND CONCLUSIONS ...................... iv Page 28 42 44 45 46 46 46 49 49 50 so 57 65 69 72 73 77 91 95 LIST OF TABLES Table Page 1 Volumes of Buffer Homogenized with Plant Material for SOD Extraction ...................... 25 2 Relationship of V/v with SOD Concentration in Crude Extracts ...................... 30 3 Relationship between V/v, b[SOD], and % Inhibition ...... 3T 4 Superoxide Dismutase Content of Seeds and Seed Parts ..... 34 5 Superoxide Dismutase Content of lO-day-old Seedlings ..... 36 6 Relative Mobility and Occurrence of Various Superoxide Dismutases in Corn, Oats, and Peas ............ 39 7 Purification of SOD from 500 g of Pea Seeds ......... SI 8 Purification of SOD from l82 g of Corn Seedlings ....... 51 9 Seedling SOD as Percentage of Hater-soluble Protein ..... 59 IO Effect of Potassium Cyanide and Chloroform-ethanol Treatments on SOD Activity of Crude Extracts ....... 59 ll Effect of Heating in a Boiling Water Bath on the SOD and Peroxidase Activity of Crude Extracts ......... 62 12 Growth, Hater-soluble Protein, and SOD Activity of Plumules ......................... 65 I3 Effect of Herbicides on the Photoreduction of NBT in the Presence of Riboflavin and Methionine ......... 79 I4 Conversion of Diuron to Hater-soluble Products by Various 02" Generating Systems .............. 86 Figure \DCDNO‘ l0 ll 12 l3 14 LIST OF FIGURES Inhibition of the NBT photoreduction versus concentration of crude SOD from corn seeds ....... Superoxide dismutases of corn, peas, and oats ....... Densitometer tracings of gels showing the seed superoxide dismutases .................. Densitometer tracings of gels showing the seedling superoxide dismutases .................. Sensitivity to cyanide and chloroform-ethanol treatments, and organelle specificity of the superoxide dismutases .................. Chromatography of pea SOD on Sephadex G-lOO ........ Chromatography of pea SOD on Bio-gel P-3O (2nd) ...... Chromatography of corn SOD on Sephadex G-TOO ........ Superoxide dismutase-like activity of horse radish peroxidase ....................... Electrophoretic comparison of superoxide dismutases with peroxidases .................... Development of SOD activity with growth and water-soluble protein content of excised pea plumules. . Development of SOD activity during germination of oats and peas ...................... Effect of paraquat on the reduction of NBT by 02 generated from xanthine/xanthine oxidase ........ Reduction of paraquat by xanthine/xanthine oxidase as indicated by the spectral change under anaerobic conditions ....................... vi Page 29 32 35 38 41 53 54 55 6T 64 67 68 80 Bl Figure Page l5 Radioactivity remaining in the water phase after extraction with methylene chloride as a function of the illumination time ................ 83 I6 Inhibition of the formation of water-soluble products from diuron by SOD ................... 84 I7 Formation of water-soluble products from diuron and inhibition by SOD as a function of pH .......... 85 l8 Radioactive products from the monuron/O " interaction and unchanged monuron recovered in the methylene chloride phase ..................... 88 19 Radioactive products from the monuron/02" interaction recovered in the water phase .............. 89 vii INTRODUCTION INTRODUCTION It is generally accepted that herbicides kill plants by inhibiting fundamental processes such as photosynthesis, respiration, and protein and nucleic acid synthesis (6)1. A simple inhibition of such processes, how- ever, does not explain some important phases of the herbicidal action (5,44,53). Inhibition of these processes may not be the real factor causing death, although it may be the primary site of action of herbi- cides. The mechanism of action of herbicides, therefore, is ambiguous, and yet the practical interest it presents has abruptly increased in recent years along with efforts to obtain safer and more effective pesticides. It has been suggested that free radicals may be involved in deteriorative mechanisms in living organisms (23). Furthermore, some herbicides have been postulated to act through generation of free radicals (5,15). Recent research supports the view that free radicals may be toxic and encourages the hypothesis that they are involved in herbicidal action. The superoxide and hydroxyl free radical are of particular interest because they can be easily formed from oxygen during various reactions of biological significance (7,35,36,40,4l), and they cause toxicity to organisms (25,26,32). Furthermore, the discovery of the enzyme 1 References at the end of "Literature review". superoxide dismutase (37) has facilitated the study of the involvement of these radicals in biological processes. Most of the information about superoxide radical and superoxide dismutase has been obtained from studies on animals and lower plants. This study was primarily designed to examine the occurrence of super- oxide dismutase in higher plants, and the quantitative differences between species. Possible involvement of superoxide radical, hydroxyl radical, and superoxide dismutase in herbicidal action was also examined by in vitro experiments. LITERATURE REV I EN LITERATURE REVIEW THE SUPEROXIDE RADICAL (02") The superoxide free radical (02") has been known to chemists for several decades (27), but it has only recently become of interest to biologists. In I954 biologists became interested in free radicals after Gerschman's group proposed that oxygen toxicity and radiation injury were due to the high reactivity of free radical intermediates (23). However, the involvement of 02" in oxidations of biological significance was not anticipated. McCord and Fridovich first proved that 02" is involved in the oxidation of sulfite and the reduction of cytochrome c and subsequently discovered the enzyme superoxide dismutase (36,37) indicating that 02" is a possible intermediate in biological systems. Currently, a considerable amount of research is being conducted in this area as indicated in a number of recent review papers (lO,l9,21,28). Only topics pertinent to this study are reviewed here. Forms of Oxygen. Taube describes the electronic structure and excited states of molecular oxygen (54). Two of his terms are to be used often in this study, namely triplet (ground) oxygen (02) and singlet oxygen (02*). The first is the normal oxygen, the lowest energy and reactivity state of oxygen. The second is one of the excited states of oxygen, particularly the state of the highest energy and reactivity, and is produced from ground oxygen after an energy input. This latter form can be toxic to organisms (l0). Triplet oxygen may also become 02" of hydroxyl radical (OH’) after univalent and trivalent reduction, respectively (2l). Superoxide radical formed from triplet oxygen further initiate production of both singlet oxygen and hydroxyl radical. Hydroxyl radical is the most potent oxidant known (43) and, therefore, its toxicity can be easily inferred. Generation of 01::. Superoxide radical is the product of univalent reduction of triplet oxygen. Divalent, trivalent and tetravalent reduction will yield H202, OH', and H20, respectively (2l). In biological systems, many enzymes catalyze the overall tetravalent reduction of oxygen to water without the release of reactive intermediates. However, the electronic structure of oxygen favors univalent pathways of reduction (54) and so generation of 02" during biological reduction of oxygen is possible. Research of recent years has provided support for this view. There is good evidence that 02" is produced during the aerobic action of several enzymes, among which are xanthine oxidase (36), numer- ous flavin dehydrogenases (35), and NADPH-cytochrome 0 reductase (7). Superoxide radical is also formed during autoxidation of a variety of compounds found in biological systems including ferredoxin (40), flavins and quinones (41), and haemoglobin (42). Generation of 02'- by isolated chloroplasts upon illumination has been reported (l,2,l2,l4). The production of 02" by leucocytes in mammals has also been established (8). Properties of 09;:, Properties of 02'- that are interesting from the viewpoint of this study are: (a). Superoxide radical can act either as a reducing or as an oxidizing agent. In the first case it gives up its extra electron and becomes 02, eg: Cytochrome c (Fe3+) + 02 + Cytochrome c (Fe2+) + 02 In the second case it becomes H202, eg: Ascorbate + 202" + 2H+ <+ Dehydroascorbate + 2H202 This latter reaction may be responsible for the effects of ascorbate on 02 uptake by isolated chloroplasts (l4). (b). In the absence of anything else for 02" to react with, it reacts with itself giving singlet oxygen (3T): .- .- + * 2 + 02 + 2H +' H202+ 02 (c). In the presence of H202, 02" produces the far more reactive 0 hydroxyl radical (27): 02' + H202 + OH + OH' + 02 (d). Compared with other oxygen radicals, 02'- is rather unreactive with a lifetime in the msec range. Biological Significance of 09;:, In vivo formation of 02’- may lead to cell damage. This can be brought about by its direct reaction with the cell components or by the generation of OH’ and 02*. Two addi- tional facts support this statement: I) the long lifetime of 02" allows it to difuse away from the site of formation (l5) and, 2) hydrogen peroxide,which accumulates in a system producing and dismutating 02" (37),subsequently leads to the production of OH' (27) capable of attacking any of the organic substances found in cells (43). There is considerable evidence that peroxidation of membrane lipids causing loss of integrity of the membrane and inactivation of membrane bound enzymes involves 02" (l0,20). Exposure of membranes to sources of 02" causes peroxidation (l6,l7,45,46,59). Bacteria and viruses exposed to 02" were rapidly destroyed (25.26.32). Superoxide radical-induced inactivation of ribonuclease and of lysine tRNA ligase was reported (32). Production of 02‘- may be responsible for the nerve degeneration caused by injecting animals with 6-hydroxydopamine (29), and 02" produced by phagocytizing leucocytes may be responsible for the degra- dation of synovial fluid and inflammation in humans (39). Reports on beneficial effects of 02" such as turnover of cell constituents and drug metabolism (22), as well as involvement in the bactericidal activity of leucocytes (8), however, suggest that 02" may also be a useful cell metabolite. SUPEROXIDE DISMUTASES (SOD) Fridovich defines superoxide dismutases as metallo-proteins that catalyze with extraordinary catalytic efficiency the reaction: .- .- + 02 +02 +2H + H202 2 is, the dismutation of superoxide radical to H202 and triplet oxygen (20). + 0 Although these proteins were known for many years as cupreins and manganins, their function was not known until I969 when the involvement of 02" in biological oxidations was substantiated (36,37). These proteins have now facilitated insight into areas unsuspected before. Aspects relevant to this study, such as occurrence, distribution, forms, and biological role of SOD are discussed below. Occurrence. SOD has been found in a wide range of aerobic organ- isms. It has been purified and characterized from bovwne erythrocytes, equine liver, bovine brain, human brain, human erythrocvtes, human and Chicken liver, bovine heart, Neurospora crassa, Fusariwn oxysporum, yeast, pea seeds, Spinach leaves, and wheat germ (20). It has also been purified and characterized from prokaryotes, mainly Escherichia coli (30) and Streptococcus mutans (55). Various aerobes that were examined were found to contain fairly fixed amounts of SOD. Aerotolerant anaerobes also contain SOD, although slightly less. However, no SOD could be detected in strict anaerobes. This led to the conclusion that SOD is of general occurrence to all aerobic organisms and constitutes the main mechanism of defense against oxygen toxicity (38). Types and Isozymes. The existence of 3 distinct types of SOD is well documented. They resemble each other in enzymatic activity, but they are different in metal content, structure, and in a number of other properties (20). 2+ and two Zn2+ atoms (a). Cupro-zinc enzymes contain two Cu per molecule, have a molecular weight around 32,000, and are composed of two subunits of equal size joined by non-covalent bonds. (b). Manganoenzymes contain two Mg2+ atoms per molecule, have a molecular weight of 80,000, and are composed of four equal subunits non-covalently joined. (c). Ferrienzymes contain two Fe3+ atoms per molecule and are characterized by a molecular weight of 39,000 and two equal subunits. Similar cupro-zinc superoxide dismutases have been isolated from a wide range of eukaryotes, but have not been found in any prokaryotes (20). Cupro-zinc superoxide dismutases are all sensitive to cyanide and this has been used as a quick test to distinguish them from the rest of the superoxide dismutases (9,56). Isozymes of cupro-zinc SOD have been reported. The enzyme from cytosol of chicken liver was resolved by disc electrophoresis into a family (four or more) of isozymes (56); also two distinct isozymes of cupro-zinc 500 were isolated and characterized from wheat germ (9). Asada and co-workers have purified to a crystalline state a single form of Cu-Zn $00 from spinach leaves (3); they further found that spinach chloroplasts contain the same enzyme, and that 30-50% of the chloro- plastic $00 was bound to the lamellar structure. They could determine little or no activity in mitochondria and no activity at all in per- oxisomes. The existence of the same enzyme in both the stroma and Iamellaecflispinach chloroplasts was recently confirmed by Elstner and Heupelusing a different assay (l3). Lumsden and Hall, however, reported on the presence of two distinct SOD enzymes in spinach leaves (33,34). One of them, which was a Cu-Zn SOD, occurred in the stroma of isolated chloroplasts. The second one, which was described as a "cyanide- resistant SOD-like activity associated with manganese", occurred in lamellae (33,34). A single Cu-Zn SOD has also been purified from dry seeds of green peas (ST). The manganese type enzyme has been found primarily in prokaryotes. In eukaryotes, it is restricted to mitochondria (20). Thus, mitochondria from chicken liver contain a Mn-SOD similar to that from bacteria; cytosol lacks this enzyme and contains only Cu-Zn enzyme (56). It was soon revealed that mitochondria contain Mn-enzyme in the matrix and Cu-Zn enzyme in the intermembrane space (57). Wheat germ contains a cyanide- resistant $00 which may be mitochondrial manganese enzyme (9). The presence of manganese enzyme in spinach chloroplasts has not been established (l3,l4). Iron containing enzyme has been found in the periplasmic space of Escherichia coli (25). Also, the blue-green alga Spirulina platensis has been reported to contain a similar ferrienzyme (33). Ferrienzyme has been reported to be the major form of soluble SOD in the blue-green alga Plectonema boryanum (4). There has been no report of ferrienzyme in higher plants. Isozymes of manganese and iron enzymes, although likely to exist, have not been observed. Induction. Oxygen has been found to induce SOD activity in organ- isms like Escherichia coli and Streptococcus faecalis (25). Thus, a 16-fold increase of SOD in Streptococcus faecalis has been achieved by raising the oxygen pressure from 0 to 20 atmospheres. In Escherichia coli, a 25-fold increase of $00 was observed when the oxygen pressure increased from 0 to 5 atmospheres. This induced SOD activity was a response to oxygen rather than to pressure, since 20 atmospheres of nitrogen had no effect. The induction was rapid with half of the maximal level reached within 90 minutes after the transfer of the cultures from anaerobic conditions to 20 atmospheres of oxygen. Induction of $00 by 85% oxygen occurs in rats (11). This has been referred to as a mechanism of acclimatization of rats by 85% O2 to resist at 100% 02. Further investigation of the $00 induction in Escherichia coli revealed that the manganese enzyme and not the ferrienzyme was induced by oxygen. The level of the latter could be changed by altering the iron supply in the medium (25). From the same studies, it was shown that the manganese enzyme serves to counter the toxicity of endogenous 02". The ferrienzyme functions as a defense against exogenous 02" There is no information yet concerning the induction of the Cu-Zn l0 enzyme. In tissues rich in Cu-Zn enzymes (eg. plant tissue), induction of manganese enzyme can be masked by the Cu-Zn enzyme, if the latter is not inducible. Biological Role. The deleterious effects that can be brought about by 02" have been demonstrated in a variety of systems. Superoxide dis- mutase provides protection in all these cases and according to Fridovich the function of the enzyme in organisms is to prevent the accumulation of this radical (19,21). Distribution studies of the enzyme are in agree- ment with this conclusion (38). Catalases and peroxidases decompose H202 and prevent production of OH' from 02" and H202, and thus, they are also essential in the overall defense mechanism against 02 toxicity (21,28). Antioxidants, such as the tocopherols and ascorbate, may function as a second line defense in scavenging these radicals (20). The possibility that 02" may also be utilized by the organism in its defense against undesirable exogenous factors has not been considered, although such cases have already been reported (8,22). Therefore, another possible role for $00 may be to regulate the concentration of a useful metabolite. Neser and co-workers recently proved that $00 also inhibits reactions induced by 02* (58). Singlet oxygen arising from 02‘- may be responsible for the effects attributed to 02" (10). This prompted the above research- ers to propose that the biological role of 500 may be protection from * 02 rather than 02 ll SUPEROXIDE RADICAL AND HERBICIDES Ashton and Crafts define the mechanism of action of herbicides as "biochemical and biophysical responses of the plant that appear to be associated with the herbicidal action" (6). According to the same authors, "the primary biochemical site of action (lesion) is the single enzyme or metabolic reaction that is affected at a concentration lower than any other enzyme or metabolic reaction, or the first reaction affected at a given low concentration". They further point out that the primary site of action may not be the answer to why a chemical is a herbicide, particularly because it may not be of such an importance to the plant that its inhibition will cause death. Frear and Shimabukuro concluded that control or death of a weed may result from one or a few key processes or, in contrast, it may require the sum of injury at several sites to attain a threshold level where total injury is irreversible and complete loss of competitive ability or death results (l8). Others have concluded specifically for inhibitors of photosynthesis that, although photosynthesis is the primary site of action, accumulation of toxic intermediates following the blockage of electron transport is the real factor causing death (5,53). The above statements indicate that effects of herbicides other than the "primary lesion" may be important in causing death. There is the possibility that formation of the toxic oxygen radicals or abolishment of the delicate defense machinery of the plant against these radicals is associated with some of these unexplained effects. There are justifi- cations for believing that this can be possible. l2 Superoxide radical is easily formed. Electron transport reactions as well as enzymatic reactions can lead to fbrmation of 02" radical. Bipyridylium herbicides become reduced to the bipyridilium radicals by the electron flow of photosynthesis and are then autoxidized to produce 02" (15). Other photosynthesis inhibitors blocking the electron transport may lead to accumulation of the reduced form of intermediate electron car- riers which when autoxidized may form 02 It is known that reduced ferredoxin (40), flavins and quinones (41) all form 02" upon autoxidation. Good proposed that deleterious oxidative processes may be involved in the killing by photosynthesis inhibitors (24). Oorschot recently provided experimental evidence to support a herbicide-induced photooxidation of the chloroplast (44). He observed that exposure of beans to COz-free air and treatment with simetone caused similar symptoms. Exposure to COZ-free nitrogen, however, delayed the onset of injury. The nature of the accumulated oxidant is not known. Isolated chloroplasts produce 02 upon illumination (2), probably by the electron flow driven reduction and subsequent autoxidation of a quinone or ferredoxin. The 02’- production in illuminated chloroplasts was suppressed when diuron was included in an assay mixture containing epinephrine as 02" detector (2). Although this indicates that the electron transport is involved in the production of 02 , it does not exclude the possibility that diuron induces 02 production at another site, perhaps the pigment system of PS II. In this case, endogenous scavengers close to that site, eg. carotenoids, may efficiently scavenge 02" making its detection with epinephrine impossible. In support of l3 this hypothesis it has been shown that addition of diuron to isolated chlorOplasts causes a fast degradation of carotenoids and only two hours later degradation of chlorOphyll begins (52). It is possible that enzymatic reactions, when disrupted by herbi- cides, proceed through alternate pathways generating 02" Superoxide radical may cause pigment degradation and destruction of membranes. Rensen showed that diquat in the light causes lipid peroxidation and decrease of chlorophyll content of Scenedesmus (50). Diuron, the antioxidant cysteine, or nitrogen flushing all strongly sup- pressed the diquat-induced lipid peroxidation providing evidence that oxygen radicals were involved. The decrease of chlorOphyll was also lessened by diuron and nitrogen flushing but to a smaller extent. The defense mechanism against 02" is very efficient but no defense is perfect (21). The existence of many forms of superoxide dismutase with differences in subcellular localization and probably function, as well as the inducibility of some forms of the enzyme, may permit the prediction that important differences between species exist. These dif- ferences then may be a factor for the selectivity of some herbicides. Frear and Shimabukuro showed that the enzyme glutathione-SLtransferase catalyzes the detoxification of s-triazines in certain plant species. They expressed the view that a major factor responsible for herbicide selectivity may be a difference in the activities, specificities, and distribution of key enzyme systems (18). Involvement of 02" or its decay products (02*, OH') in biological transformations of herbicides is also possible. Superoxide radical is both a reducing and an oxidizing agent (28), and the OH’ is one of the 14 most potent oxidants (43). Evidence for involvement of 02" in sulfoxi- dation of thioethers was obtained with ethionamide (49). Hydroxylation of aromatic compounds catalyzed by enzymes from AspergiZZus niger was shown to involve 02" (48). s-Triazine herbicides were dealkylated by an OH‘-generating system to products identical to those isolated from various biological systems (47). LITERATURE CITED 1. ALLEN, J.F. AND 0.0. HALL. 1973. Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chlorOplasts. Biochem. BiOphys. Res. Commun. 52:856-862. 2. ASADA, K. AND K. 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Preparation and physicochemical properties of green pea superoxide dismutase. Biochim. Biophys. Acta 268:305-312. STANGER, C.E., JR. AND A.P. APPLEBY. 1972. A proposed mechanism for diuron-induced phytotoxicity. Weed Sci. 20:357-363. SWEETSER, P.B. AND C.W. TODD. 1961. The effect of monuron on oxygen liberation in photosynthesis. Biochim. Biophys. Acta. 51:504-508. TAUBE, H. 1965. Oxygen: chemistry, structure and excited states. Little, Brown and Co. Boston. 55. 56. 57. 59. 20 VANCE, P.G., B.B. KEELE, JR., AND K.V. RAJAGOPALAN. 1972. Superoxide dismutase from Streptococcus mutans: isolation and characterization of two forms of the enzyme. J. Biol. Chem. 247:4782-4786. WEISIGER, R.A. AND I. FRIDOVICH. 1973. Superoxide dismutase: organelle specificity. J. Biol. Chem. 248:3582-3592. WEISIGER, R.A. AND I. FRIDOVICH. 1973. Mitochondrial superoxide dismutase: site of synthesis and intramitochondrial localization. J. Biol. Chem. 248:4793-4796. WESER, U., W. PASCHEN, AND M. YOUNES. 1975. Singlet oxygen and superoxide dismutase (cuprein). Biochem. Biophys. Res. Commun. 66:769-777. ZIMMERMANN,R., L. FLOHE, U. WESER, AND H.J. HARTMANN. 1973. Inhibition of lipid peroxidation in isolated inner membrane of rat liver mitochondria by superoxide dismutase. FEBS-Lett. 29:117-120. SECTION ONE OCCURRENCE OF SUPEROXIDE DISMUTASES IN HIGHER PLANTS 21 OCCURRENCE OF SUPEROXIDE DISMUTASES IN HIGHER PLANTS ABSTRACT The photochemical assay for superoxide dismutase consisting of methionine/riboflavin/p-nitro blue tetrazolium chloride was adjusted for quantitation of the enzyme in crude extracts. Shoots, roots, and seeds of corn (Zea mays L., cv. Michigan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were analyzed for their superoxide dismutase content. The enzyme is not organ specific. It is present in the shoots, roots and seeds of all three species. Quan- titative and qualitative differences exist between species, while quanti- tative differences exist between organs within a species. On a per dry weight basis, shoots contain more enzyme than roots. Both shoots and roots contain considerably more enzyme than seeds. In seeds, the enzyme is present in both the embryo and the storage tissue. Electrophoresis indicated multiple forms of the enzyme. From the three species, a total of 10 distinct enzyme bands was obtained on gels. Corn contained seven of the bands, oats only three. Peas contained one of the corn, and two of the oat enzymes. Nine of the enzyme bands were eliminated with cyanide treatment suggesting that they may be cupro-zinc enzymes, whereas one was cyanide resistant and may be a manganese enzyme. Some of the superoxide dismutases were primarily found in mitochondria or chloroplasts. 22 23 Differences and similarities in SOD pattern of the various organs may be explained on basis of the above organelle localization of the S00 forms. INTRODUCTION Superoxide dismutases (EC 1.14.1.1) are metalloproteins catalyzing the reaction: 2 T H202 i.e. the dismutation of the superoxide free radical (02") to molecular 202” + 2H+ + 0 oxygen and hydrogen peroxide. This enzymatic activity was first described by McCord and Fridovich with a cupro-zinc protein (erythrocuprein) from bovine erythrocytes (5). Similar cupro-zinc proteins with SOD1 activity were subsequently isolated from various eukariotic sources (8). Manganese- containing proteins with SOD activity were later found in prokaryotes and in mitochondria of eukaryotes (8). Iron proteins from Escherichia coli (10) and algae (13) were recently shown to possess SOD activity. Cupro-zinc SOD has already been isolated from tissues of higher plants: pea seeds (17), spinach leaves (1,13), and wheat germ (4). Isozymes have also been reported for wheat and spinach cupro-zinc enzyme (4,13). A cyanide resistant enzyme from wheat germ has been described as manganese- SOD (4). Occurrence of manganese enzyme in spinach chloroplasts has been reported (13), but is not certain (7). There is no report of iron-enzyme in higher plants. There is considerable evidence that 02" may be an indigenous inter- mediate of metabolic processes which may initiate deteriorative effects 1 Abbreviations: SOD: superoxide dismutase; NBT: p-nitro blue tetrazolium chloride. 24 in biological systems; $00 is believed to constitute an important part of the defense mechanism against such deleterious action of 02" (9,11). A comparative study of the S00 distribution in plants has not been conducted thus far. In view of the above evidence, however, distribution of the enzyme within the plant and intraspecific differences may be of interest, and were the objective of the present study. MATERIALS AND METHODS Plant Material. Seeds and seedlings of corn (Zea mays L., cv. Mich- igan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were utilized. Seeds were treated with 0.3% (w/v) captan 80W for 5 min and germinated in plastic flats containing 3 cm turface (Turf Supplies Co., Taylor, Michigan) at the bottom and 3 cm vermiculite at the top. The seedlings were grown for 10 days in a growth chamber under 26 C, 25000 lux of light, and a 12-hr photoperiod. Half-strength Hoagland's solution was provided once after six days, and distilled water throughout growth as needed. Embryos and scutella were excised from seeds which had been allowed to imbibe at 25 C for 10 hr and were rinsed three times with distilled water. Endosperm was obtained from dry corn and oat seeds from which the embryo-bearing end had been removed. Endosperm was drilled from the cut surface by using a size 60 wire bit fitted on a battery operated drill. The hulls of oat seeds were removed, unless the seeds were to be germi- nated. I Preparation of Extracts. The tissues were thoroughly ground with a cold mortar and pestle in an ice bath, until no fibrous residue could be 25 seen. The grinding medium consisted of 0.1 M potassium phosphate and 0.1 mM EDTA, pH 7.8, plus homogenizing glass beads. The homogenate was centrifuged twice at l3000g for 10 min in a Sorvall RC2-B refrigerated centrifuge at O to 5 C. The supernatant, hereafter referred to as crude SOD extract, was used for electrophoresis and for determination of the $00 content in the tissue. Different volumes of buffer were used for enzyme extraction, depen- ding on the tissue and whether the extract was to be used for electropho- resis or determination of the S00 content (Table 1). For determination Table 1. Volumes of’Buffer Homogenized with Plant Material for SOD Extraction. Extracts for Extracts for 500 Plant Part Species Electrophoresis Determination Leaves, Roots, Shoots All species 4 m1/g fresh wt 10 ml/g fresh wt Dry seeds Corn 6 m1/g seed wt 15 ml/lO seeds Dry seeds Oats 6 m1/g seed wt 10 m1/10 seeds Dry seeds Peas 6 ml/g seed wt 30 m1/10 seeds Soaked seeds All species 4 ml/g wet wt same as dry seeds of the $00 content in the tissue, the volumes necessary for quantitative extraction of the enzyme were predetermined. The water-soluble protein content of all crude SOD extracts was determined by the method of Lowry et al. (12), after precipitation with 10% trichloroacetic acid. Bovine serum albumin was used as a standard. Extracts were diluted 10 times for SOD assays. To the extracts for electrophoresis l M sucrose was added. 26 Isolation of Organelles. Mitochondria were isolated by the method of Bonner (5) and chloroplasts by the method of Smillie (19) from shoots of 7-day-old seedlings grown in the dark and light, respectively. The organelle pellets were washed twice by resuspending in wash media and recentrifuging. The washed pellets were finally resuspended in small volumes of 0.05 M potassium phosphate, pH 7.8, and sonicated for 3 min. After dialysis against changes of the same buffer and centrifugation, the supernatants were used for electrophoresis. ElectrOphoresis. Polyacrylamide gel electrophoresis of the crude SOD extracts was performed according to Davies (6). Each extract was applied to the gels at various concentrations ranging from 50 to 300 ug of protein, or 5 to 20 units of enzyme. A current of 1 mA/gel was applied during migration of the bromphenol blue marker on the spacer gel. Electrophoresis was continued under 2 mA/gel until the marker had migrated approximately 10 cm on the resolving gel. The superoxide dismutases were localized by the photochemical pro- cedure of Beauchamp and Fridovich (3) as modified by Weisiger and Fridovich (20). The gels were first soaked in a solution of NBT, then immersed in a solution of riboflavin and tetramethylenediamine, and i1- luminated in tubes containing potassium phosphate and EDTA at pH 7.8. The stained gels were photographed on Kodak Panatomic X 120 film using a 25 A red filter, or scanned with a Gilford model 220 gel densitometer. Enzyme Assay. All extracts were assayed for SOD activity photo- chemically, using the assay system consisting of methionine, riboflavin, and NBT (3). The photochemical procedure was chosen as being independent of other enzymes and proteins, and therefore more reliable in the case of crude extracts than enzymatic assay systems (15). 27 The original assay described by Beauchamp and Fridovich (3) was modified. The reaction mixture was composed of 1.3 pM riboflavin, 13 mM methionine, 63 uM NBT, 0.05 M sodium carbonate pH 10.2, and the appropriate volume of extract. Distilled water was added to bring to the final volume of 3 ml. The mixtures were illuminated in glass test tubes selected for uniform thickness and color. Identical solutions that were not illuminated served as blanks. The apparatus devised for exposing the tubes to light was composed of a rotating test tube holder (Rayonet MGR-100, Southern New England UV Co.) immersed in water in a cylindrical glass container thermostated at 25 C by a Forma 2095 refrigerated and heated water circulator. A circular fluores- cent lamp (Sylvania, FC 12 T lO-CW-RS) was attached on the outside wall of the water-bath and the entire assembly was fitted in a box lined with aluminum foil. The reaction was initiated and terminated by turning on and off the light. There was no detectable amount of the reaction occur- ring under room light during preparation of the solutions and spectrophoto- metric measurements. The initial rate of the reaction was determined as increase of absorbance at 560 nm. Under the described conditions, the initial rate of the reaction in absence of $00 was 0.100 absorbance units/5 min and was linear up to 15 min. In the presence of $00 the reaction was inhibi- ted and the amount of inhibition was used to quantitate the enzyme. Each extract was assayed twice and the results varied less than 10.005 absorbance units/5 min. Dialysis and gel filtration (Sephadex G-50) of crude SOD extracts indicated no significant interference of small molecules with the assay. 28 RESULTS AND DISCUSSION Enzyme Quantitation. The assay system used in this study utilizes the photochemical production of 02'- from methionine, riboflavin, and oxygen, and the subsequent reduction of NBT to blue formazan. Superoxide dismutase, by scavenging the 02", inhibits the photoreduction of NBT. Beauchamp and Fridovich defined one unit of $00 as the amount that inhibits the NBT photoreduction by 50% and quantitated the enzyme on basis of the % inhibition it causes (3). Percent inhibition and $00 concentration were not linear, however. Asada et a2. (2), using crystalline spinach SOD and the xanthine/xanthine oxidase assay system (15), established a linear relation- ship between the amount of the enzyme and the V/v ratio (V,v represent the rate of the assay reaction in absence and in presence of SOD, respectively). This linear relationship was also observed with crude corn SOD and the photochemical assay system used in the present study (Fig. l). Asada et al. also derived from the kinetics of the assay reaction the equation: V/v = 1 + K'[SOD] (I), which explains the linearity obtained. Asada's plot was further tested in this study with a variety of crude extracts, and the above equation was properly adapted for convenient and accurate quantitation of $00. The linearity between V/v ratio and $00 concentration was maintained throughout a wide range of enzyme concentrations (Table 2). The upper limit of the linear portion varied with the various extracts in the range of 67 to 84% inhibition. The linear correlation was high, as indicated by correlation coefficients close to unity. The maximum inhibition of the NBT photoreduction that could be achieved by the crude SOD used was 93 to 97%. The reason linearity is not maintained up to this maximum 29 6 o o——o on O I L D z o _ b I 2, E60 6 El . E40 '- :096‘* .1 4 : A92 d - > $20 _ 5 e ‘1 _ 2 V 4 l I l l I oo 20 40 so so 1oo"JO SOD CONCENTRATION (pl crude extract/3 ml) Fig. 1. Inhibition of the NBT photoreduction versus concentration of crude $00 from corn seeds. Crude extract prepared from 10 corn seeds homogenized with 15 ml of 0.1 M potassium phosphate, pH 7.8, was diluted various times with the same buffer and each dilution was used at 100 ul/3 ml assay-solution to give the indicated SOD concentrations. Percent inhibition is not linear with $00 concentration (o-o). Linearity (r = 0.995) is obtained by plotting V/v ratio against SOD concentration (De—4>), V and v representing the'rate of the reaction in absence and presence of enzyme, respectively. 30 is not known. Table 2. Relationship of V/v with SOD Concentration in Crude Extracts. Crude extracts were diluted and assayed to obtain the V/v versus [SOD] curve as in Figure I. The apparent linear portion was determined between the lower limit (always 0%) and the upper limit (the maximum % inhibition up to which the linear relationship was attained). Regression analysis of the X,y pairs within the linear portion (at least 6 pairs) was conducted to obtain the line equation and the correlation coefficient. Extract Upper Limit Correlation Species Part (% inhibition) Line Equation Coefficient (r) Corn Seeds 84 y=1.04 + 0.054X 0.995 Oats Seeds 68 y=1.02 + 0.140X 0.994 Peas Seeds 67 y=1.08 + 0.047X 0.998 Oats Roots 69 y=1.09 + 0.045X 0.997 Peas Roots 67 y=1.06 + 0.041X 0.986 Oats Shoots 73 y=0.96 + 0.317X 0.997 Peas Shoots 70 y=0.96 + 0.058X 0.994 The intercept of all lines obtained from the various extracts (Table 2) approximates unity, this being in agreement with equation (I) of Asada et al. The slope (b) of the lines, corresponding to the constant K' in equation (I), varies with the various extracts as they are different in $00 content, and fits the equation: y - l = bX or (V/v) - l = b [SOD] (II) One unit of SOD has been defined as the amount of enzyme that causes 50% inhibition of the assay reaction (3,15). The relationship between V/v,b[SOD], and % inhibition of the NBT photoreduction is illustrated in Table 3. Apparently, V/v and b[SOD] are linearly related. One SOD unit 31 Table 3. Relationship between V/v, b[SOD], and % Inhibition. V/vl b[SOD]2 % Inhibition3 l 0 0.0 2 I 50.0 3 2 66.6 4 3 75.0 1 V = rate of the NBT photoreduction in absence of SOD (uninhibited reaction); v = rate of the NBT photoreduction in presence of S00 (inhibited reaction). 2 b[SOD] = (V/v) - 1 (see text). 3 % Inhibition = [(v-v)/vl 100. can be defined as the amount that either causes 50% inhibition or gives a product b[SOD] equal to unity. If the latter definition is adopted, advantage can be taken of the more linear curve of Asada et al., and further, the enzyme can be directly quantitated from the V/v ratio according to the equation: 500 units/ml = [(V/v) - 1] (dilution factor) (III). Superoxide Dismutase Content of Plant Organs and Tissues. Botn enzyme assays and electr0ph0resis established that extractable $00 was present in seeds and various seed parts, roots, leaves and shoots of all three species. Electrophoresis also indicated that SOD activity is composed of a number of distinct bands. Within a species, the same active bands with apparent quantitative differences were obtained from seeds, roots and shoots (Fig. 2). From the different species, a different number of bands were obtained. In corn, six bands were visually observed, in contrast to only three in oats and 32 Fig. 2. Superoxide dismutases of corn, peas, and oats. Extracts from seeds (left gel in each group), leaves (middle gels), and roots (right gels) were applied for each species. The enzymes appeared as achromatic bands on the blue-stained gels. The negative image used here, in which the bands appear black, shows their positions more clearly. The corn $00 was resolved into at least six visually observable bands, where- as that of peas was resolved into two, and that of oats into three major bands (numbers on the left). 33 two in peas. Only bands of high relative mobility were obtained from oats and peas, whereas corn also contained bands of intermediate and low relative mobilities (Fig. 2). High concentrations of oat and pea extracts did not produce additional bands, even though the observed bands overlapped. The corn bands of intermediate and low relative mobil- ity were also observed at considerably lower concentrations of extract. It may be concluded that the above differences in band numbers are real and not due to concentration effect. The same banding patterns were obtained under various extraction conditions, such as pH 6 to 8, potassium phosphate concentrations 0.005 to 0.4 M, and presence or absence of 0.1 mM EDTA. Dialysis and ammonium sulfate or acetone fractionation of the crude extracts did not alter the banding patterns. However, freeze- thawing and aging in the cold resulted in additional minor bands. In seeds, $00 was present in both the embryo and the storage tissue (Table 4). In this study the units of enzyme per seed were determined photochemically from extracts of whole seeds, embryos and storage tissue. It was also established that during imbibition of seeds from O to 25 hr, the total activity of enzyme per seed as well as the activity per embryo and storage tissue were not altered. Evidence is provided that a fixed amount of SOD is always present in the embryo and the storage tissue and no de novo synthesis or activation needs to occur upon imbibition. Most of the enzyme in oat and pea seeds was found in the storage tissue; the oat endosperm containing 62% and the pea cotyledons 82% of the total activity (Table 4). By contrast, in the corn seed, more enzyme was found in the embryo than in the storage tissue. This may be attributed to the fact that the corn embryo (including the scutellum) constitutes a considerably larger portion of the whole seed than the oat or pea embryo. 34 Table 4. Superoxide Dismutase Content of'Seeds and Seed Parts. Each value is the average of 8 non-significantly different values obtained from seeds soaked in water for 0, 5, 10, and 25 hr (2 samples each). Species Whole Seedl Embryo2 Storage Tissue3 SOD units/seed Corn 40.7 26.7 17.7 Oats 20.5 8.0 12.7 Peas 792.2 142.0 652.0 SOD units/g dry wt Corn 194 1057 96 Oats 884 4819 565 Peas 4681 39776 3739 1 Purification of the enzyme from pea seeds did not indicate any interference of impurities with the crude extract assays (Table 7). 2 The scutellum of corn and oats is included in the embryo. 3 Storage tissue: the remaining part of the seed after removal of the embryo. On a per dry weight basis, the embryo of all three species contains approx- imately 10 times more enzyme than the storage tissue. Therefore, the embryo is richer in SOD regardless of containing more or less enzyme than the storage tissue on a per seed basis. The occurrence of the enzyme in both the embryo and the storage tis- sue was further confirmed by electrophoresis (Fig. 3). The active bands were obtained from the embryo of all three species. Scutella excised from the corn seeds contained the same SOD bands as the corn embryo. No attempt was made to demonstrate the presence of enzyme in scutella of 35 .25 A 7 A 1 ’ 1 .20 ' o o I a .15" i- 1 ' ' JO" " " ’ ‘ a 0 mam ‘ scum mm .olelll‘blll LILLlLlll llllLlL E c O o in in 0 z ‘ In «um um» C 141 llJlL IlILllll lllllll 2‘05 J o 2 4 a a 10 .25 2 c c M30 .20:- :- AIOORN '15" F mono owns .10” 1' m— r- L‘” ”It: 0 LILIJ ‘ lllllLll O 2 4 6 8 10 O 2 4 6 8 10 mm M1410 Fig. 3. Densitometer tracings of gels showing the seed superoxide dismutases. Extracts from the indicated seed parts were used at 10 ul per gel, except for the extracts from oat and corn endosperm, which were used at 50 and 100 01 respectively. The enzymes, which appeared as achromatic bands on blue-stained gels, are shown here as negative peaks, and are numbered in order of increasing relative mobility. Rm: relative mobility (migration distance of band/migration distance of marker). 36 oats. Endosperm drilled out of dry corn and oat seeds, and cotyledons excised from dry pea seeds contained the same SOD bands as their embryos. Quantitative differences between the $00 bands will be discussed in the next section. The occurrence of the enzyme in all seed parts, and more importantly in the endosperm is of particular interest. If the proposed biological role for this enzyme (9,11) is accepted, then its distribution may demonstrate that 02" can be formed in seeds, and may be involved in aging and reduced viability of seeds, as suggested by Pammenter et al. (16). The enzyme was found in both shoots and roots of the seedlings (Table 5). In this study the seedlings were grown up to the stage that the shoots Table 5. Superoxide Dismutase Content of'lO-day-old Seedlingsl. Each value is the average of two samples extracted and assayed independently. Dry wt Protein Units/ Units/mg Units/mg Species Organ (mg/plant) (mg/g dry wt) plant dry wt protein Corn Shoot 143 57.5 579.1 4.0 69.5 Corn Root 146 20.8 124.1 0.9 41.3 Oats Shoot 13 132.8 89.7 6.9 51.9 Oats Root 13 32.2 31.8 2.5 76.2 Peas Shoot 67 208.4 304.8 4.6 22.0 Peas Root 62 51.2 127.1 2.0 20.0 LSD at 0.05 ... 18.6 206.5 1.7 19.6 1 The enzyme has been purified from pea seeds and corn seedlings and interferences with the assay are negligible for crude extracts from seed- lings of the three species (see section two). 37 and roots of each species accumulated the same amount of dry matter at harvest. On a per dry weight basis, the shoots of all three species con- tain more protein and S00 than the roots. There is no statistically significant difference between $00 content of shoots and roots of peas on a per protein basis. The most important difference among the three species is that the least SOD-active protein is present in roots for corn, in shoots for oats,and in both shoots and roots for peas. The enzyme of roots is compared with that of leaves in Figure 4. Differences in quantities of enzyme on a per protein basis can be judged from the area below the bands. The two grass species, particularly corn, contain more enzyme in the roots. Peas contain more enzyme in the leaves. Simon et al. (18) also found more enzyme in leaves than in roots of beans. Multiple Forms of SOD. Ten distinct SOD bands were obtained from extracts of the three plant species in a single electr0ph0retic run. They are numbered in order of increasing relative mobility in Figures 3 and 4, and summarized in Table 6. It has also been concluded from their relative mobilities that $00 6 and S00 8 are common to peas and oats, while $00 9 is common to peas and corn. In two different plant species, therefore, some S00 forms may be identical, and some may be different. The above 10 bands may correspond to different proteins with SOD activity, or different isozymes of a single protein, or a combination of both. Proteins containing copper-zinc, manganese, or iron have already been described to function as superoxide dismutases (8). Isozymes of cupro-zinc SOD have also been reported (4,13). Cupro-zinc enzymes are sensitive to cyanide, whereas manganoenzymes are resistant to cyanide and sensitive to treatment with a chloroform- 38 I I I I L 1 5'2“? one o f o .. ”I . h u a (z) .150- . F < O czjoot ~ 0 2 0001 LIA? no ( I I L LI 1 I I I I I I I I I I L ’ peas ...... 16F i f o .150:- - 100- U - u . 1‘ 000! an . I I I L I L LIL LI 4 I I JJ 1 I 0 2 ‘4 6 8 K) 0 2 ‘4 6 8 I) RHINO BmlIO Fig. 4. Densitometer tracings of gels showing the seedling super- oxide dismutases. Extracts from roots and leaves were used at 50 ug of protein per gel, except for the extract from corn leaves, which was used at 100 ug of protein. The enzymes, which appeared as achromatic bands on blue-stained gels, are shown here as negative peaks, and are numbered in order of increasing relative mobility. Rm: relative mobility (see Fig. 3). 39 ethanol mixture, thus allowing their discrimination on gels (4,20). The only enzyme that was found resistant to cyanide was $00 5 of corn (Fig. 5). It seems likely, therefore, that SOD 5 is a manganoenzyme, all the remain- ing being isozymes of cupro-zinc SOD. Table 6. Relative Mobility and Occurrence of'Various Superoxide Dismutases in Corn, Oats, and Peas. SOD Band1 Relative Mobility2 Occurrence l 0.08 - 0.09 corn 2 0.18 - 0.25 corn 3 0.41 - 0.42 corn 4 0.52 - 0.54 corn 5 0.61 - 0.62 corn 6 0.65 - 0.67 corn,peas 7 0.69 - 0,70 corn 8 0.74 - 0.75 oats,peas 9 0.78 - 0.80 corn,peas 10 0.87 - 0.91 oats 1 Numbers correspond to the $00 bands as in Figures 3 and 4. 2 Defined as in Figure 3. The same S00 enzymes were obtained from the various organs or tissues within a species (Figs. 3 and 4), and this supports the reproducibility of the bands. However, quantitative differences of the bands among organs or tissues are evident from the "band area". The following differences between seed parts can be seen in Figure 3. Corn endosperm does not contain any detectable amount of $00 1, and com- pared to the other seed parts is lower in 500 3 and 4, but higher in S00 2. 40 A similar situation occurs in oat seeds; the endosperm is low in $00 6 and high in $00 8, exactly the opposite of the embryo. The remaining part of the oat seed after the removal of the embryo, which includes aleurone and endosperm, is high in both S00 6 and 8 suggesting that the aleurone must be similar to the embryo. There is no significant differ- ence between pea embryo and cotyledons. The localization of some of the $00 enzymes in organelles was ex- ploted as a possible explanation of the above differences. Mitochondria were isolated from etiolated seedlings and the extracts from them were subjected to electrophoresis at various concentrations. Results are presented in Figure 5. The $00 enzymes in which endosperm is rich (S00 2 and 8) were not detected in mitochondrial extracts. The $00 enzymes in which embryo, aleurone, or cotyledons are rich (SOD 1,3,4, and 6) were major bands in mitochondria. Therefore, a possible expla- nation for the above differences may be the fact that $00 1,3,4, and 6 are primarily, but not exclusively, localized in mitochondria. Embryo, scutellum, aleurone, and cotyledon tissue contains a large number of mitochondria, in contrast to the endosperm. Major similarities between the S00 pattern of the roots (Fig. 4) and that of the embryos (Fig. 3) are in agreement with the localization of certain SOD forms in mitochondria. The leaves, compared to both seeds and roots, present the major difference that $00 9, or 10, is the major band in them. Chloroplasts isolated from leaves of young seedlings con- tained more of these two enzymes than mitochondria (Fig. 5). This dif- ference in $00 pattern between mitochondria and chloroplasts may be the reason for the difference in $00 pattern between leaves and roots. 41 1. 12 £3 4: 5 IS a 2:3 E E Beam 5” ' / ;/ . D OATS B.‘ 8/ 10” w r PEAS a... “T 0 Fig. 5. Sensitivity to cyanide and chloroform-ethanol treatments, and organelle specificity of the superoxide dismutases. Treatments (numbers at the tap) are as follows: (1) leaf extract, (2) leaf extract and cyanide treatment, (3) leaf extract and chloroform-ethanol treatment, (4) mitochondrial extract, (5) chloroplastic extract. Potassium cyanide at 1 mM was included in the staining solutions for treatment (2). For treatment (3), the extracts were mixed with chloro- form (0.15 volume) and ethanol (0.25 volume), and centrifuged before applied on the gels. The enzyme bands are numbered (on the left) as in Figures 3 and 4. 10. 42 LITERATURE CITED ASADA, K., M. URANO, AND M. TAKAHASHI. 1973. Subcellular location of superoxide dismutase in spinach leaves and preparation and properties of crystalline spinach superoxide dismutase. Eur. J. Biochem. 36:257-266. ASADA, K., M. TAKAHASHI. AND M. NAGATE. I974. Assay and inhibitors of spinach superoxide dismutase. Agr. Biol. Chem. 38:471-473. BEAUCHAMP, C.0. AND I. FRIDOVICH. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276-287. BEAUCHAMP, C.O. AND I. FRIDOVICH. 1973. Isozymes of superoxide dismutases from wheat germ. Biochim. Biophys. Acta 317250-64. BONNER, W.D., Jr. 1965. Mitochondria and electron transport. In J. Bonner and J.E. Varner, eds., Plant Biochemistry. Academic Press, New York, pp. 89-123. DAVIES, B.J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427. ELSTNER, E.F. AND A. HEUPEL. 1975. Lamellar superoxide dismutase of isolated chloroplasts. Planta 123:145-154. FRIDOVICH, I. 1974. Superoxide dismutases. Adv. Enzymol. 41:35-97. FRIDOVICH, I. 1975. Oxygen: boon and bane. Amer. Scientist 63:54-59. GREGORY, E.M., F.J. YOST, AND I. FRIDOVICH. 1973. Superoxide dismutases of Escherichia coli: intracellular localization and functions. J. Bacteriol. 115:987-991. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 43 HALLIWELL, B. 1974. Superoxide dismutase, catalase and glutathione peroxidase: solutions to the problem of living with oxygen. New Phytol. 73:1075-1086. LOWRY, O.H., N.J. ROSEBROUCH, A.L. FARR, AND R.J. RANDALL. 1951. Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193:265-267. LUMSDEN, J. AND D.O. HALL. 1974. Soluble and membrane-bound superoxide dismutases in a blue-green alga (Spirulina) and spinach. Biochem. Biophys. Res. Commun. 58:35-41. LUMSDEN, J. AND 0.0. HALL. 1975. Chloroplast manganese and superoxide. Biochem. Biophys. Res. Commun. 64:595-602. MC CORD, J.M. AND I. FRIDOVICH. 1969. Superoxide dismutase - an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244:6059-6055. PAMMENTER, N.W., J.H. ADAMSON, AND P. BERJAK. 1974. Viability of stored seed: extension by cathodic protection. Science 186:1123-1124. SAWADA, Y., T. OHYAMA, AND I. YAMAZAKI. 1972. Preparation and physicochemical properties of green pea superoxide dismutase. Biochim. Biophys. Acta 268:305-312. SIMON, L.M., Z. FATRAI, D.E. JONAS, AND B. MATKOVICS. 1974. Study of peroxide metabolism enzymes during the development of Phaseolus vulgaris. Biochem. Physiol. Pflanzen 166:387-392. SMILLIE, R.H. 1972. Synthesizing capability of the photosynthetic apparatus: proteins. Methods in Enzymology 24:381-394. WEISIGER, R.A. AND I. FRIDOVICH. 1973. Superoxide dismutase: organelle specificity. J. Biol. Chem. 248:3582-3592. SECTION TWO PURIFICATION AND PROPERTIES OF SUPEROXIDE DISMUTASE FROM HIGHER PLANTS 44 PURIFICATION AND PROPERTIES OF SUPEROXIDE DISMUTASE FROM HIGHER PLANTS ABSTRACT Superoxide dismutase was purified to a maximum specific activity from pea (Pisum sativum L., cv. Wando) seeds, and partially purified from corn (Zea mays L., cv. Michigan 500) seedlings. ihe purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase. It was estimated that in lO-day-old seedlings of corn, peas and oats (Avena sativa L., cv. Au Sable) 0.9 to 3.1% of the water soluble protein is accounted for by superoxide dismutase. Interferences with the enzyme assays of crude extracts were examined. Peroxidase at high concentrations interferes with the assay. Peroxidase was easily inacti- vated by heat, whereas superoxide dismutase was relatively heat-stable. In crude extracts from seedlings of all three species the interference by peroxidase was negligible. A 3-fold increase of specific activity 45 46 (units/mg water-soluble protein) was observed during germination of oats, and a 40% increase during greening and hook opening of the pea plumule. INTRODUCTION In a previous study, considerable amounts of SOD1 were found to be present in roots, shoots, seeds and seed parts of oats, corn and peas (see section one). Electrophoresis indicated multiple forms of the enzyme. Significant differences in quantity and forms of the enzyme were observed between species and between organs within a species. The objective of this study was to further substantiate the occurrence of the enzyme in higher plants and to examine the observed differences between species. For this purpose the enzyme was purified. Impurities interfering with the assays of crude extracts, and changes of SOD specific activity during seedling growth were also studied. MATERIALS AND METHODS Enzyme Purification. Unless otherwise stated all operations were performed at 0 to 4 C. Dry pea seeds (Pisum sativum L., cv. Wando) were soaked in distilled water for about 15 hr. The resulting 1650 g wet weight was crushed with an electric mortar and pestle, and homogenized with 1 liter of 0.1 M KZHPO4 in a Waring blender. After stirring, the slurry was filtered squeezed through six layers of cheese-cloth. The 1 Abbreviations: SOD: superoxide dismutase; NBT: p-nitro blue tetrazolium chloride. 47 filtrate was centrifuged twice at I3000g for 30 min in a Sorvall RCZ-B refrigerated centrifuge. The supernatant was subjected to the Tsuchihashi (chloroform-ethanol) treatment essentially as described by Weisiger and Fridovich (13). It was established that none of the pea SOD enzymes is inactivated by this treatment. The supernatant was mixed with 0.25 volume of ethanol and 0.15 volume of chloroform and stirred for 15 min. It was then clarified by centrifugation at 130009 for 15 min. Chloroform that was separated out during centrifugation was removed by suction. The supernatant was decanted, solid KZHPO4 (20 g/liter) was added, and the two phases were separated after 30 min. The less dense, ethanol rich phase was collected, chilled and centrifuged at -15 C. Additional chloroform separating out during centrifugation was removed by suction, and the ethanolic phase was decanted. Chilled acetone (0.5 volume) was added to the ethanolic phase while stirring. The precipitate was removed by centrifugation. Additional acetone (1.5 volume) was added to the supernatant, and the second precipi- tate was collected and redissolved in a minimal volume of 0.05 M potassium phosphate buffer pH 7.8. Solid (NH4)2S04 was added to the supernatant to bring it to 70% saturation. After 1 hr, the second precipitate was col- lected, and resuspended in and dialyzed against 0.1 M KC1, 0.005 M potassium phosphate, and 0.01 mM EDTA, pH 7.8. The enzyme was further purified by gel exclusion chromatography. It was first applied on a Sephadex G-100 column (2 x 90 cm) equilibrated with the dialysis buffer. The void volume of the column was 74.5 ml and the flow rate 0.2 mI/min. This column was calibrated with proteins of known mol wt (11). Fractions with a specific activity greater than 48 300 units/mg protein were pooled and salted out from 70% saturated (NH4)2SO4 solution. The precipitate was collected by centrifugation, dissolved in a small volume of distilled water and dialyzed against the eluting buffer. It was then applied on a column (1 x 60 cm) of Biogel P-30 equilibrated with the same buffer. Some impurities of higher mol wt were removed by this column. Fractions whose specific activity exceeded 700 units/mg were pooled and concentrated as above. The enzyme was rechromatographed on the same Biogel P-30 column. Impurities of slightly lower mol wt were partially separated from the enzyme. Only the two fractions of maximum specific activity (around 2000 units/mg) were pooled this time. Additional impurities were removed by fraction- ation with chilled acetone. The most active fraction was obtained between 1.5 to 2.0 volumes of acetone. The precipitate from the last fraction was redissolved in 0.05 M potassium phosphate, pH 7.8. Enzyme from corn (Zea mays L., cv. Michigan 500) seedlings was partially purified. The seeds were treated with 0.3% (w/v) captan 80W for 5 min, and germinated for 7 days on moist paper towels in the dark at room temperature. The seed remnants were removed, and the seedlings (182 g) were rinsed with distilled water and cut into l-cm sections with a stainless steel razor blade. The tissue was homogenized with 400 ml of 0.1 M potassium phosphate and 0.1 mM EDTA, pH 7.8, in a Waring blender. After 1 hr in the cold and occasional stirring, the homogenate was fil- tered squeezed through four layers of cheese-cloth. The filtrate was centrifuged twice at 130009 for 20 min. This supernatant was not sub- jected to the Tsuchihashi treatment, since it has been shown that this treatment inactivates one of the corn enzymes (Fig. 5). The enzyme was successively purified by acetone fractionation (0.75 to 2.0 volumes), 49 (NH4)2SO4 fractionation (45 to 95% saturation) and chromatography on Sephadex G-100 in a manner similar to the pea enzyme. Protein concentration was determined throughout according to Lowry et al. (9), using bovine serum albumin as a standard. Enzyme assays and electrophoresis were performed as previous described. Interference Experiments. Crude extracts used in these experiments were prepared from shoots and roots of lO-day-old seedlings as previously described. Catalase (beef liver, Nutritional Biochemicals Corporation) proved to be contaminated with $00, and required purification by gel exclusion chromatography. Horse radish peroxidase (A grade, Calbiochem) was not contaminated with $00. Peroxidase activity was determined with the guaiacol test (12). Gels were stained for peroxidase localization according to Hart et al. (7). Crude extracts were heated in portions of 1.0 ml in Korex l7-ml centrifuge tubes kept in a boiling water bath (95 to 97 C) for 0.5 to 20 min. Precipitated protein was removed by centrifugation. Changes of SOD Specific Activity. The changes in specific activity during greening were studied with excised oat (Avena sativa L., cv. Au Sable) and pea plumules. Seeds, treated with 0.3% (w/v) captan 80W for 5 min, were soaked in distilled water for 10 hr and planted 2-cm deep in 10 x 14.5 cm styrofoam pots containing vermiculite. Seedlings were grown in a growth chamber in complete darkness at 25 C for 7 days. Plu- mules were excised by cross-secting with a stainless steel razor blade above the first node from the apex. Uniform plumules were transferred into 9-cm petri dishes (10 plumules/dish) containing 10 m1 of 1% sucrose solution. Three replicate dishes for each treatment were prepared. Half of the dishes were placed in a growth chamber with all lights off. 50 The other half were placed in another growth chamber with only the fluor- escent lights on (10 and 7 uW/cm2 blue and red light, respectively). The temperature in both chambers was maintained at 25 C. At various time in- tervals, petri dishes were removed from the chambers, the plumules were rinsed with distilled water, blotted and weighed. Extracts were prepared and the assays were performed immediately. All operations with etiolated plumules were conducted under dim green safelights. The changes - C) 100:! *2 s - —i .2- 80 .< u: 0 A E 460 S m z: m (D O \ (0.1 ' ‘40 3 00 ~23 4: ~20 O t - O 10 20 30 4O 50 60 7O FRACTION NUMBER Fig. 7. Chromatography of pea S00 on Bio-gel P-30 (2nd). The column (1x60 cm) was equilibrated and the enzyme eluted with the same buffer described in Figure 6. The enzyme collected from a first run on this column was concentrated to 0.5 ml, dialyzed, and rechromato- graphed. 55 )- '7 o—o Absorbance E .6» o-—o Actwnty o u 33 .5- E m .4~ 420) <9 C) 3 -_i m .3- 9° 5 GE -4 8 2 < In F A < 5 1- -30 g :3. r- L 20 56—‘6—‘4 50 60 H0 FRACTION NUMBER Fig. 8. Chromatography of corn $00 on Sephadex G-100. The column and buffer were the same described in Figure 6. The 45 to 95 % (NHu)2SOu precipitate was redissolved in and dialyzed against the buffer before loading the column. Fractions are 4.0 ml each. 56 minor peak represents a polymeric form of the enzyme, and that aging in the cold promoted polymerization. During the course of this study, stor- age of pea or corn enzyme resulted in additional faint bands on gels. This indicates that the plant enzyme polymerizes similarly to the chicken enzyme. The polymerization apparently does not inactivate the enzyme. The major peaks eluted from the Sephadex column represent the bulk of the enzyme which did not undergo any alteration during purification. The enzyme from each species eluting as the major peak from Sephadex is apparently homogeneous with regard to mol wt, heterogeneous with regard to electrophoretic properties. This supports the view that the S00 bands correspond to isozymes of $00. The major peak obtained with pea enzyme' contained all three SOD bands found in the crude extract. These bands could be eliminated with cyanide, indicating they are due to isozymes of cupro-zinc SOD. The major peak obtained with corn enzyme contained five out of the seven SOD bands found in the crude extract. All of these bands could be eliminated with cyanide and, thus, are isozymes of cupro-zinc SOD. Pea and corn cupro-zinc enzyme have the same mol wt (approximately 30000) as indicated by similar elution volumes from the Sephadex columns (Figs. 6 and 8). The two 500 bands of corn that were not present in the major peak were $00 2 and 5. The $00 2 band was a cyanide-sensitive protein and was shown to be an artifact due to peroxidase (Fig. 10). The $00 5 band was a cyanide resistant, chloroform-ethanol sensitive protein, which may be a manganese-containing SOD. The manganese enzyme has a considerably higher mol wt than the cupro-zinc enzyme (4), and therefore, it would not copurify with the major peak. The enzyme from oats was not purified. It was shown, however, that 57 one of the three SOD bands of oats was an artifact due to peroxidase (Fig. 10). The other two bands had the same relative mobility on gels as two of the pea bands, and therefore, they may also correspond to isozymes of cupro-zinc SOD. Seedling Enzyme as Percentage of the Water-soluble Protein. The specific activity in photochemical units/mg protein was found to be 14 and 2039 for the crude and the purified pea enzyme, respectively (Table 7). The photochemical unit as defined in the previous study is equivalent to 3.03 standard units (10). Therefore, the specific activity in standard units/mg protein is 42 and 6178 for the crude and the purified pea enzyme, respectively. All specific activities in the following discussion are in standard units/mg protein. The enzyme from pea seeds was also purified by Sawada et al. (11). They determined a specific activity for the pure enzyme of 6400, which is similar to the specific activity found in this study. However, their specific activity for the crude enzyme was 9.9, considerably lower than determined in this study. Sawada et al. used the xanthine/xanthine oxidase assay system (10) for their assays. Evidence was obtained in the course of this study that impurities in crude extracts depress the enzyme activity when determined by this assay system (see section on interferences). This may explain the low specific activity of the crude enzyme as determined by Sawada et al. The purified pea enzyme was tested for purity by electrophoresis. One weak band not corresponding to any SOD-active protein was localized on the gels. The actual specific activity of the enzyme, therefore, is expected to be somewhat higher than the above value. Asada et al. have purified spinach leaf $00 to a crystalline state (2). The specific 58 activity of this enzyme was 9320. Their method of protein determination (absorption at 258 nm) was different from the Lowry procedure used in this study and by Sawada et al., thus, a direct comparison of the values may not be relevant. However, it is reasonable to assume that the specific activity of SOD in higher plant species is within the range of 6178 to 9320. The specific activity of the crude enzyme in roots and shoots of seed- lings has already been determined (Table 5). This data was used to esti- mate the percentage of water-soluble protein accounted for by $00, assuming specific activities for the pure enzyme in the range 6178 to 9320 (Table 9). Superoxide dismutase accounts for 0.9 to 3.1% of the water-soluble protein. Interferences with Enzyme Activity in Crude Extract Assays. Experi- ments were conducted to study interference of impurities in the crude extracts with the enzyme activity. Thus, a basis could be provided for accessing the reliability of the data presented in the previous section. The effect of chemical treatments on the SOD activity of crude extracts was studied. Complete loss of activity was achieved, when 3 mM KCN was included in the assay mixture (Table 10). Chloroform-ethanol treatment of the extracts caused an approximately 5% loss of activity only in the case of corn. Cupro-zinc $00 is inactivated by cyanide and manganese $00 by the chloroform-ethanol treatment (13). Even though com- plete loss of activity was obtained with potassium cyanide, other enzymes may interfere with the assay, since cyanide is not a Specific inhibitor of SOD. However, the results from this experiment indicate that there is no interference by small mol wt impurities. Dialysis of the crude extracts confirmed this conclusion. Catalase and peroxidase are other enzymes which might interfere with the photochemical assay. Catalase at concentration as high as Table 9. 59 Seedling SOD as Percentage of Water-soluble Protein. Species Part SOD (% of water-soluble protein) Minimum1 Maximum2 Average Corn Shoot 2.3 3.4 2.85 Corn Root 1.3 2.0 1.65 Oats Shoot 1.7 2.5 2.10 Oats Root 2.5 3.7 3.10 Peas Shoot 0.7 1.1 0.90 Peas Root 1.3 2.0 1.65 1 Assuming specific activity for $00 9320 units/mg protein, as reported for enzyme purified to crystalline state from spinach leaves (2 2 Assuming specific activity for S00 6178 units/mg protein, as deter- mined in the present study for pea seed enzyme. Table 10. Effect of Potassium Cyanide and Chloroform-ethanol Treatments on SOD Activity of Crude Extracts. SOD Activity % Extract Treatment (units/ml) Inhibition Corn None 50.0 ... Corn Chloroform-ethanol 42.6 5.4 Corn KCN 1 mM 10.0 80.0 Corn KCN 3 mM 0.0 100.0 Oats None 62.3 ... Oats Chloroform-ethanol 62.1 0.0 Oats KCN 3 mM 0.0 100.0 Peas None 31.5 ... Peas Chloroform-ethanol 31.5 0.0 Peas KCN 3 mM 0.0 100.0 60 150 09/3 ml did not mimic SOD in retarding the accumulation of blue formazan. Horse radish peroxidase, however, did retard the accumulation of blue formazan, thus exhibiting an SOD-like activity (Fig. 9). This activity was equivalent to 40.6 photochemical units/mg of peroxidase and was not due to contamination of this enzyme with $00. In the photochemical assay system used in this study, SOD inhibits the reduction of NBT to blue formazan. Peroxidase is not likely to inhibit the reduction of NBT but is likely to catalyze the oxidation of the blue formazan formed. Hydrogen peroxide, which is required for the action of the peroxidase, is produced by the assay system. In absence of SOD, H202 is produced at low concen- trations from the spontaneous dismutation of 02" (8). Higher concentra- tions of H202 are produced in the presence of $00 from both spontaneous and enzymatic dismutation of 02" (10). When purified pea SOD and horse radish peroxidase were both included in the assay mixture, the SOD-like activity of the peroxidase was doubled (Fig. 9). Increasing the S00 concentration did not further increase the activity of the peroxidase. This suggests that low H202 concentration was limiting the action of peroxidase in absence but not in the presence of SOD. Attempts were made to estimate the extent to which peroxidase in the crude extracts could interfere with the S00 assays (Table 11). Per- oxidase and $00 activities were both determined in crude extracts of corn, oats and peas. Peroxidase activity varied from 263 to 9022 units/m1. As estimated from Figure 9, peroxidase at these concentrations would account for less than 2.5 500 units/ml, which is probably not statistically significant. It was further observed that peroxidase could be easily inactivated by heating the crude extracts in a boiling water bath; $00 was relatively heat-stable. The peroxidase of the crude extracts was 61 Apparent SOD Units PEROXIDASE (units x163) Fig. 9. Superoxide dismutase-like activity of horse radish peroxi- dase. The photochemical assay system for S00 was used. Reaction mix- tures contained the indicated amounts of peroxidase (H), or the indicated amounts of peroxidase plus 0.7 unit of purified pea SOD (o—o). The synergistic effect of 500 on the SOD-like activity of peroxidase is apparent by the difference in the slope of the predicted (o—---) and observed (o—o) lines. 62 completely inactivated after heating for 1.5 min. Inactivation of the peroxidase was not accompanied by any loss of SOD activity in the corn and oat extracts. Some loss of SOD activity which was observed in the case of the pea extract may not be due to the inactivation of peroxidase, since pea SOD seems to be less heat-stable than corn and oat enzyme. After heating for 20 min, corn, oat and pea extracts retained 100, 30 and 20% of the SOD activity respectively. It may be concluded from these results that interference by peroxidase is negligible, and corrections in Table 9 need not be made. Table 11. Effect of’Heating in a Boiling water Bath on the SOD and Peroxidase Activity of’Crude Extracts. Heating SOD Activity Peroxidase Activity Species Time (min) (units/m1) (units/ml) Corn 0.0 65.7 263 Corn 1.5 69.0 00 Corn 20.0 66.1 Oats 0.0 67.0 3579 Oats 1.5 66.8 0 Oats 3.0 52.0 Oats 20.0 19.8 Peas 0.0 21.0 9022 Peas 0.5 19.5 6315 Peas 1.5 17.0 0 Peas 20.0 4.1 63 The heat-stability of $00, which was observed when the crude extracts were heated, was not observed when purified enzyme was heated. Corn and pea SOD partially purified with (NH4)ZSO4 and acetone fraction- ation were completely inactivated by heating at 75 C for 2 to 3 min. Ad- dition of corn crude extract to the purified corn and pea enzyme before heating provided partial protection. These results may suggest that impurities in the crude extracts, and especially in the corn crude extract, protect $00 from heat inactivation. In the case of corn crude extract, this may also explain the resistance of the enzyme to precipitation by (NH4)2504 or acetone (Table 8). In another experiment, purified pea enzyme was mixed with crude extracts at various proportions and assayed. The SOD activity of the mixtures was equal to the sum of the activities, when the photochemical assay system was used; but, it was less than the sum of the activities, when the xanthine/xanthine oxidase assay system (10) was used. This observation provides additional evidence that the photochemical assay system is reliable for the determination of SOD concentration in crude extracts. Electrophoresis resolves the $00 of crude extracts into a number of dintinct bands. The possibility that some of these bands may be due to peroxidase was examined (Fig. 10). Two out of 10 $00 bands obtained from the three species, namely S00 2 of corn and $00 10 of oats, coin- cided with peroxidase bands indicating that these two bands may be artifacts due to peroxidase. This also may be the reason purified corn enzyme does not contain the S00 2 protein. It is evident that peroxidase interfered more during electrophoresis than during assays. Localization of $00 on gels was performed at pH 7.8, whereas assays were at pH 10.2. corn 0 I. 1 [I 2- 2 I (3) I (I) '~4 (II ~48 (33 Li A B 64 oats A D B peas __, it A B Fig. 10. Electrophoretic comparison of superoxide dismutases with peroxidases. Crude extracts from shoots of lO-day-old seedlings were applied on gels. For each species, one gel was stained for S00 (gels A), and one gel for peroxidase (gels B). numbered as before (Figs. 3 and 4). Superoxide dismutase bands are 65 The optimum pH for peroxidase is 7.0 (3), which may explain the above observation. Changes in Specific Activity of SOD. Evidence for the inducibility of SOD has been presented for bacteria (5,6) and blue-green algae (1). In this study, 500 specific activity was shown to increase during green- ing of pea plumules and germination of oats. Pea and oat plumules excised from seedlings grown in the dark were either kept in the dark or transferred to the light. After 48 hr, the $00 specific activity of the green plumules was compared with that of the etiolated ones (Table 12). Green pea plumules had developed a specific Table 12. Growth, water-soluble Protein, and SOD Activity of'Plumules. Plumules were excised from 7-day-old seedlings grown in the dark, placed in petri dishes containing 1% sucrose, and kept at 25 C in the dark (E) or under cool white light (G) for 48 hr. Species Etiolated (E) Fresh wt SOD (units/ Protein (mg/ SOD units/ or Green (G) (mg/plumule) g fresh wt) 9 fresh wt) mg protein Oats E 36.5 712.0 12.2 59.3 Oats G 36.8 992.7* 16.9* 58.7 Peas E 39.6 883.7 23.9 37.4 Peas G 67.0* 901.0 17.3* 52.2* * F value for difference between the means of the species is significant at the 0.05 level. 66 activity which was approximately 40% higher than that in the etiolated pea plumules. Green and etiolated oat plumules had developed the same specific activity. A different growth response to light by the two species seems to be associated with these results. Over the 48-hr period, pea plumules grew faster in the light (hook opening, plumular expansion) than in the dark. Oat plumules grew at the same rate in the dark and light. On a fresh weight basis, green oat plumules accumulated more $00 than etiolated oat plumules; but they also accumulated propor- tionally more water-soluble protein and thus the specific activity did not increase. Associated with increased growth rate of pea plumules in the light is a decrease of water-soluble protein but not of $00. This explains the increased specific activity of pea plumules with greening. Results from a kinetic experiment with pea plumules indicated that the increase of S00 preceded that of growth (Fig. 11). Although, on a per plumule basis, water-soluble protein decreased with time at the same rate both in the dark and light, SOD increased. However, the increase of $00 was faster in the light. Growth in the light was accelerated after a lag period of 12 hr. The rapid increase in $00 preceded the acceleration of growth. The development of the $00 specific activity in oats and peas was also examined during the first days of termination (Fig. 12). On the first day, the specific activity was approximately the same in oats and peas. The specific activity in oats rapidly increased and remained at a high level. In peas, it remained at the initial low level. From experi- ments with lO-day-old seedlings, it was observed that the $00 specific activity was considerably lower in peas than in oats. The above results confirm this observation and indicate that the difference between the two 67 73 S E 3 'a \ 1’3. '5 3 0 TI 0 R 09 CD I 20_ growths-,0 5 0L _ L" 6051’ :1 3 I509 A U .42 0 E :325L 2 2 _ £3 E 3201- Z - DJ 15- 5 . E10 1 - I I I ' O 12 24 48 TIME IN LIGHT OR DARK (hf) Fig. 11. Development of SOD activity with growth and water-soluble protein content of excised pea plumules. Plumules were excised from 7-day-old seedlings grown in the dark, placed in petri dishes containing 1 % sucrose solution and kept in either dark or light conditions. 68 32"." $00 oats 0:4 Fkoufin 824' 5 Peas E 3 3‘16 .2 r «1233 C a peas '3: C) (MB-- 887 ts TE /"08 . . L . S O 0 5 E €60.- WIS U E \ (D 2240" C 3 8 " I I I I O 1 2 3 4 AGE (days) Fig. 12. Deve10pment of SOD activity during germination of oats and peas. Seeds were germinated in the dark, on moist filter paper in petri dishes. 69 species is established since early during germination. The physiological significance of the above described changes in $00 specific activity cannot be assessed at the present time. These changes, however, may support the reality of previously described varia- tions in SOD content from organ to organ and species to species. LITERATURE CITED 1. ABELIOVICH, A., D. KELLENBERG, AND M. SHILO. 1974. Effect of photooxidative conditions on levels of superoxide dismutase in Anacystis nidulans. Photochem. Photobiol. 19:379-383. 2. ASADA, K., M. URANO, AND M. TAKAHASHI. 1973. Subcellular location of superoxide dismutase in spinach leaves and preparation and properties of crystalline spinach superoxide dismutase. Eur. J. Biochem. 36:257-266. 3. CHANCE, B. AND A.C. MAEHLY. I955. Assay of catalases and peroxidases. Methods in Enzymology 2:764-776. 4. FRIDOVICH, I. 1974. Superoxide dismutases. Adv. Enzymol. 41:35-97. 5. GREGORY, E.M. AND I. FRIDOVICH. 1973. Induction of superoxide dismutase by molecular oxygen. J. Bacteriol. 114:543-548. 6. GREGORY, E.M., F.J. YOST, Jr., AND I. FRIDOVICH. 1973. Superoxide dismutases of Escherichia coli: intracellular localization and functions. J. Bacteriol. 115:987-991. 7. HART, M.A., H. TYSON, AND R. BLOOMBERG. 1971. Measurement of activity of peroxidase isozymes in flax (Linum usitatissimum). Can. J. Botany 49:2129-2137. 10. ll. 12. 13. 70 KHAN, A.V. 1970. Singlet molecular oxygen from superoxide anion and sensitized fluorescence of organic molecules. Science 168:476-477. LOWRY, O.H., N.J. ROSEBROUGH, A.L. FARR, AND R.J. RANDALL. 1951. Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193:265-267. MC CORD, J.M. AND I. FRIDOVICH. 1969. Superoxide dismutase - an enzymatic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244:6049-6055. SAWADA, Y., T. OHYAMA, AND I. YAMAZAKI. 1971. Preparation and physicochemical properties of green pea superoxide dismutase. Biochim. Biophys. Acta 268:305-312. SIMON, L.M., Z. FATRAI, D.E. JONAS, AND B. MATKOVICS. 1974. Study of peroxide metabolism enzymes during the development of Phaseolus vulgaris. Biochem. Physiol. Pflanzen 166:387-392. WEISIGER, R.A. AND I. FRIDOVICH. 1973. Superoxide dismutase: organelle specificity. J. Biol. Chem. 248:3582-3592. SECTION THREE IN VITRO PRODUCTION OF SUPEROXIDE RADICAL FROM PARAQUAT AND ITS INTERACTION WITH MONURON AND DIURON 71 IN VITRO PRODUCTION OF SUPEROXIDE RADICAL FROM PARAQUAT AND ITS INTERACTION WITH MONURON AND DIURON ABSTRACT The ability of herbicides to produce superoxide radical as well as their ability to react with this radical was examined through their effect on the superoxide-induced reduction of p-nitro blue tetrazolium chloride. Paraquat enhanced and diuron inhibited the reduction of p-nitro blue tetrazolium chloride. Paraquat was reduced photochemically (riboflavin/methionine) or enzymatically (xanthine/xanthine oxidase) and produced superoxide radical upon reoxidation. Diuron and monuron interacted with photochemically produced superoxide radical, but not with enzymatically produced superoxide radical. The product of the monuron/superoxide interaction was a demethylated, dechlorinated water-soluble compound containing phenolic hydroxyl group(s), and was not toxic to oats. The enzyme superoxide dismutase prevented the formation of this product. Other herbicides (atrazine, metribuzin, terbacil, 2.4-0. CDEC, diphenamid) had little effect on the p-nitro blue tetrazolium chloride reduction. 72 73 INTRODUCTION The occurrence of superoxide free radical (02") in organisms is suggested by at least two independent pieces of evidence. First, the wide distribution of the enzyme S001 (14). which scavenges the free radical and is thus thought to constitute the basis of a defense mech- anism against its deleterious action (8,10). Secondly, the documented production of 02" from a variety of chemical and enzymatic reactions of biological origin. Reduced ferredoxins (15), flavins and quinones (16) produce 02" upon reoxidation. The aerobic action of xanthine oxidase (14), flavoenzymes (l3), NADPH-cytochrome c reductase (2). and other enzymes gives rise to 02 Production of 02" by illuminated chloro- plasts (l) and by leucocytes (4) has also been demonstrated. Rapid killing of cells subjected to 02" and other detrimental effects to biological systems associated with 02" or its transient decay products (singlet oxygen, hydroxyl radical) (8) has prompted in- vestigators to examine the possibility that the toxic action of certain bactericides and herbicides is due to their ability to produce 02". Steptonigrin was shown to produce 02" (23) and its toxicity to Escherichia coli could be reduced by inducing the SOD activity of the organism (9). Production of 02" from diquat (21) and paraquat (7) was also shown in vitro. Paraquat-induced toxicity to rats could be reduced by admin- istering SOD intravenously (3). The possible involvement of 02" in biological transformations of pesticides should also be considered. Superoxide radical is both a 1 Abbreviations: SOD: superoxide dismutase; NBT: p-nitro blue tetrazolium chloride; FMN: riboflavin 5'-phosphate sodium. 74 reducing and an oxidizing agent (10). and the hydroxyl radical arising from 02" is one of the most potent oxidants (8). Evidence for involve- ment of 02" in the sulfoxidation of thioethers was obtained with ethionamide (20). Hydroxylation of aromatic compounds catalyzed by enzymes from Aspergillus niger was shown to involve 02" (19). s-Triazine herbicides were dealkylated by a hydroxyl-radical generating system to products identical to those isolated from various biological systems (18). Direct proof of 02'- involvement in action or metabolism of herbi- cides from in vivo experiments is not easy to obtain at the present time due to lack of a reliable technique for detecting the radical in plant tissues. However. reliable information may be obtained from in vitro experiments utilizing model systems developed in recent years. In the present study, the ability of herbicides from various chemical classes to either produce 02‘- or to react with 02" was tested by using such model systems. It became evident from these studies that paraquat is able to produce 02". whereas monuron and diuron readily react with 02" The mechanism of 02" production from paraquat and the interaction of monuron and diuron with 02" were further studied. MATERIALS AND METHODS Non-radioactive herbicides2 were analytical grade and recrystal- lized from appropriate solvents. Carbonyl-14C-labeled monuron and diuron 2 Chemical names of herbicides: paraquat: 1,1'-dimethyl-4,4'-bipyridylium dichloride; 2.4-0: (2.4-dichlorophenoxy) acetic acid; CDEC: 2-chloroallyl diethyldithiocarbamate; diuron: 3-(3.4-dichlorophenyl)-l,l-dimethylurea; monuron: 3-(p-chlorophenyl)-l.l-dimethylurea; terbacil: 3-tert-butyl-5- chloro-6-methyluracil; atrazine: 2-chloro-4-(ethylamino)-6-(isopropyl- amino)-s-triazine; metribuzin: 4-amino-6-tert-butyl-3-(methylthio)-as- triazin-5(4H)one; diphenamid: N,N'-dimethyl-2,2'-diphenylacetamide. 75 were obtained from E.I. du Pont de Nemours and Company and purified by TLC. The Rf values of the radioactive herbicides were identical with those of authentic monuron and diuron in two solvent systems. Xanthine and xanthine oxidase (from milk) were purchased from Sigma Chemical Corporation, NBT from Aldrich Chemical Company, Inc. Catalase (from beef liver) was obtained from Nutritional Biochemicals Corporation and was chromatographed on Sephadex G-100 to remove contaminant 500. Super- oxide dismutase was purified from pea seeds and corn seedlings as des- cribed elsewhere (section two). All other chemicals were of analytical reagent grade and the solvents of pesticide grade. The systems utilized to produce 02" were based on the autoxidation of photoreduced flavins (5), the oxidation of xanthine by xanthine oxidase (14), and the autoxidation of reduced phenazine methosulfate (17). These systems were used as described in the literature, unless otherwise indicated. All experiments were conducted at room temperature. Photo- reactions were routinely performed in glass test tubes (Kimax. 1.7 x 14.5 cm) using cool white light from a circular lamp (Sylvania, FC12T lO-CW-RS) at 25 C as previously described (see section one). Ultra violet and visible absorption spectra were obtained with a Beckman DB-G grating spectrophotometer equipped with a Sargent Model SR recorder. Infra red spectra were measured with a Perkin Elmer Model 337 IR spectrophotometer and KBr discs of the compounds. Radioactivity was measured with a Packard 3003 Tri-Carb Scintillation Spectrophotometer using internal spiking with 14C-toluene to determine 0PM. Formation of water-soluble compounds from carbonyl-14C-labeled monuron and diuron were quantitatively followed by extracting the reaction mixtures with an equal volume of methylene chloride. The two 76 phases were separated after centrifugation and the radioactivity deter- mined in each phase by transferring 0.2 ml to counting vials containing 15 ml of a scintillation solution consisting of 666 m1 toluene, 333 m1 Triton-X-lOO, 4g PPO and 50 mg dimethyl-POPOP. Corrections were made for monuron and diuron remaining in the water phase under the various experimental conditions. Formation of methylene chloride-soluble com- pounds was quantitated by TLC analysis of the methylene chloride phase. Three ml of this phase were transferred into a screw-cap test tube, the solvent was evaporated under vacuum. and the residue redissolved in 0.5 m1 acetone. An 0.2-ml alquot of the acetone was spotted on pre-coated TLC plates (Silica G-25 UV254, Brinkmann Instruments. Inc.) and over- spotted with 5 pg of non-radioactive monuron or diuron. The plate was developed three times in the same direction to a 15-cm solvent front with benzene-acetone (3:1 v/v). Monuron or diuron was localized under ultra violet light and the spot carefully scraped with a razor blade and transferred into a vial containing 15 ml of scintillation liquid (600 m1 toluene. 400 ml methyl-cellosolve, 33.3 ml water, 4 g 8801 and 80 g naphthalene). Preparative separation of the products from monuron was carried out according to the following procedure. The reaction mixture consisted of 500 ml of a 50 0M solution of monuron (12300 DPM/ml) in distilled water, 2.8 mg riboflavin, 16.8 mg EDTA, 258.7 mg K HPO and 456 mg 2 4 KH2P04 (pH 6.5). The mixture was placed in a l-liter round bottom flask (Pyrex) and illuminated for 2 hr under constant mechanical stirring in a box lined with aluminum foil and equipped with a circular fluorescent lamp. The average light intensity on the outside surface of the flask was 4850 luxes. The reaction mixture was extracted twice with 500 m1 77 of methylene chloride. Sixty-two % of the original radioactivity was recovered in the water phase and 30% in the methylene chloride phase. The methylene chloride phase was dried with anhydrous calcium chloride, the solvent evaporated under vacuum, and the residue redissolved in 1.0 ml of acetone. An 0.25-ml aliquot of the acetone was chromato- graphed on a TLC plate as in the quantitative experiments. The radio- chromatogram was obtained by scraping off the gel in l-cm strips and measuring radioactivity. The water phase was freeze-dried and the residue repeatedly extracted with acetone and ethanol. After the solvents were removed by evaporation under vacuum. the residue was redissolved in 1.5 m1 of dis- tilled water. The recovery of the water-soluble radioactivity was approxi- mately 78%. This solution was applied in 0.5-ml portions on a sephadex. G-lO column (1.5 x 60 cm). The column was eluted with distilled water, fractions of 4 ml were collected and assayed for radioactivity. The fractions corresponding to the major radioactive peak (92% of total radioactivity in the effluent) were tested for purity by measuring absorbance at 230 nm. The absorption and the radioactivity peaks coin- cided. indicating that no contaminant was copurified with the major water- soluble product. The combined fractions were used in toxicity tests after dilution or in identification tests after freeze-drying. RESULTS AND DISCUSSION Effect of Herbicides on the Reduction of N81. Superoxide radical is generated during reoxidation of photoreduced riboflavin (5), or oxidation of xanthine by xanthine oxidase (14). The production of O2 78 by either system can be measured spectrophotometrically by following the reduction of NBT. since 02" reduces N81 to blue formazan (5). Infor- mation on the ability of a herbicide to produce 02" or to react with 02'- may be indirectly obtained from the effect it will have on the NBT reduction. The NBT reduction by 02'- generated from photoreduced riboflavin was measured in presence of various herbicides (Table 13). Atrazine, metribuzin, diphenamid, CDEC, 2.4-0 and terbacil had little effect on the rate of NBT reduction. Paraquat markedly enhanced and diuron markedly inhibited the N81 reduction. The effect of paraquat and diuron on the NBT reduction by the xanthine/xanthine oxidase system was also examined. Paraquat enhanced the NBT reduction by this system (Fig. 13). This enhancement could be prevented with SOD. Paraquat itself did not reduce NBT. Clearly, the effect of paraquat on N81 reduction is due to increased production of 02". Diuron, on the other hand, did not affect the NBT reduction by the xanthine/xanthine oxidase system, although it inhibited NBT reduction by photoreduced riboflavin. A possible explanation for this discrepancy is presented in the section on diuron. Production of 09" from Paraquat. It has been well documented by using isolated chloroplasts that paraquat and related compounds are capable of accepting a single electron and becoming reduced to the cor- responding free radicals (6.12.24). The xanthine/xanthine oxidase system used in this study also reduced paraquat as indicated by the characteristic spectral change under anaerobic incubation (Fig. 14). Similarly, accumu- lation of the paraquat free radical was evident from development of intense violet color upon illumination of a solution contain.ng paraquat, riboflavin. 79 Table 13. Effect of'Herbicides on the Photoreduction of’NBT in the Presence of'Riboflavin and Methionine. Reaction mixtures contained 1.5 uM riboflavin, 0.01 M methionine 60 pM NBT, and 0.1 M sodium carbonate pH 10.2. Herbicides were dissolved in ethanol (paraquat in distilled water) and added so that the final con- centration was 0.1 mM for the herbicide and 1% for the ethanol. Ethanol at 1% did not affect the NBT reduction. Rate of NBT Reduction1 Herbicide (AA56O/min) % of Control None 0.025 ... Paraquat 0.042 168 2,4-0 0.021 84 CDEC 0.030 120 Diuron 0.012 48 Terbacil 0.022 88 Atrazine 0.027 108 Metribuzin 0.028 112 Diphenamid 0.028 112 1 LSD: 0.004 at 5%, 0.006 at 1%. 80 E .200 § 6 =i £15“) I) “I Ta g .100 00 fl: 8 .050 3 ,_ 6,0 0 i l J J o 1 2 TIME (min) Fig. 13. Effect of paraquat on the reduction of NBT by 02" generated from xanthine/xanthine oxidase. The standard reaction mixture contained 0.1 mM EDTA, 20 pH xanthine, 60 pH NBT, 0.05 M potassium phosphate, pH 7.8, and 0.05 ml of a xanthine oxidase solution. Modifi- cations of the standard mixture: (a) none; (b) plus 0.3 mM paraquat; (c) plus 3 mM paraquat; (d) plus 30 units of SOD; (e) plus 3 mM paraquat and 30 units of 500; (f) minus the xanthine oxidase and plus 3 gM pgfiaquat. Changes of absorbance at 560 nm were recorded with a Sargent Mo e1 recorder. 81 L5F :" N l ABSORBANCE .0 NO I 9 Ch I oa-‘nay o} -------------------------- 320 40° 5°° 60° 70° WAVElEN'I'I-l (nm) Fig. 14. Reduction of paraquat by xanthine/xanthine oxidase as indicated by the spectral change under anaerobic conditions. The cuvette contained the standard reaction mixture (Fig. 13), except that NBT was omitted and paraquat was added at 6 mM. The cuvette was equili- brated with air ( ----- ) or nitrogen ( ). Spectra were recorded 5 min after addition of the xanthine oxidase. 82 and methionine in absence of air. Because of its low redox potential, the paraquat radical is rapidly reoxidized in presence of oxygen. It was recently shown by Farrington et al. that 02" is produced upon reoxidation of the paraquat free radical (7). The enhancement of NBT reduction by paraquat in connection with the fact that $00 prevents this enhancement are in agreement with the finding of Farrington et al. that 02 is produced from paraquat. Further support is provided by the fact that paraquat can be reduced by the two 02" gener- ating systems used in this study. The two systems and NBT can probably be used as convenient assays for testing suspected production of 02" from other pesticides. Interaction of Diuron and Monuron with 0,;:, An aqueous solution of carbonyl-14C-labeled diuron. riboflavin and methionine was exposed to light. At time intervals, aliquots were extracted with methylene chloride to remove the diuron. The amount of radioactivity in the water phase in- creased with time of illumination, indicating that diuron was converted to water-soluble compounds (Fig. 15). Superoxide dismutase inhibited the formation of water-soluble compounds by at least 70 % (Fig. 16). The yield of water-soluble compounds from diuron was maximum at pH 6.0. and the inhibition by $00 was maximum at pH 8.0 to 9.0 (Fig. 17). Experiments 14 with carbonyl- C-labeled monuron yielded the same results. Conversion of diuron and monuron to water-soluble compounds results from an inter- action of the herbicides with 02", since S00 is effective in preventing this conversion. Diuron reacted with 02" produced photochemically from flavins. but not with 02" produced from other systems (Table 14). The photochemical system was still effective in converting diuron to water-soluble compounds 83 ‘ GD 1 .n Cl 1 .e II .a I3 lb nAmoacnvnv m WATER (DPM-162/2mll 0 so 40 ILLUMINATION TIME (min) Fig. 15. Radioactivity remaining in the water phase after extraction with methylene chloride as a function of the illumination time. Reaction mixtures (final volume 4.0 ml) containing 1.5 uM riboflavin. 0.01 M methionine, 15 uM carbonyl-1“C-labe1ed diuron, and 0.1 M potassium phosphate. pH 6.0, were illuminated, extracted with 4.0 ml of methylene chloride. and the radioactivity in 0.2 ml of the water phase determined. 84 100$ X INHIBITION I I I 4‘1 0 5 1O 15 20 25 30 l l S 0 D (mute/4 ml) Fig. 16. Inhibition of the formation of water-soluble products from diuron by $00. Reaction mixtures as in Figure 15 containing the indicated amounts of S00 were illuminated for 7 min. 85 !_ 1 '- BUFFERS 0.1M 1 12_ H N- ACETATE 6&3 ir—a x PHOSPHATE .; H KCARBONATE E 10 --100 9. (D g a -80 3 E 6'- 460 u: .I g 4— -4O .1 I'.‘ < s L.,- O @1- I 5*———3——‘——é——$—‘ib7 11 ° pl! Fig. 17. Formation of water-soluble products from diuron and inhibition by 500 as a function of pH. Reaction mixtures containing riboflavin. methionine, and diuron as in Figure 15, and the indicated buffers were illuminated for 7 min. at 3.0 units/4 ml. Superoxide dismutase was added c>———«3 % INHIBITION BY SOD 86 Table 14. Conversion of’Diuron to water-soluble Products by Various 02" Generating Systems. Reaction mixtures contained in a final volume of 4 ml: carbonyl-14C- diuron 45 nmoles; riboflavin, FMN. xanthine, or phenazine methosulfate 45 nmoles; methionine, EDTA. or NADH 50 nmoles; potassium phosphate, pH 6.5, 150 umoles. Catalase was added at 30 units and $00 at 6 units. Incubation time was 1 hr. Diuron Converted to Water- 02 Generating Systems and Modifications soluble Products (nmoles/4 ml) Riboflavin + Methionine + Light 27 minus riboflavin, methionine, or light 0.0, or'O plus pea S00 10 plus boiled pea $00 28 plus corn S00 11 plus boiled corn S00 27 plus catalase 27 Riboflavin + EDTA + Light 30 minus riboflavin 0 plus pea $00 16 plus catalase 30 FMN + EDTA + Light 31 minus EDTA 0 plus pea S00 13 plus catalase 30 Xanthine + EDTA + Xanthine Oxidase 0 Phenazine methosulfate + NADH 0 87 when riboflavin was substituted by FMN and methionine by EDTA. The xanthine/xanthine oxidase (l4) and phenazine methosulfate/NADH (17) systems were not effective, although they produced 02 Conversion of diuron to water-soluble compounds was possible only when a complete photochemical system was employed, and it could be inhibited only by $00, which indicates that 02" is undoubtedly responsible for the con- version. Catalase had no effect, which excludes the possibility that hydrogen peroxide or hydroxyl radical produced from 02" are involved. Interaction of diuron with photochemically produced 02" but not with 02" produced from xanthine/xanthine oxidase may explain why diuron inhibited NBT reduction by the former but not by the latter system. Monuron has been reported to inhibit the photooxidation of EDTA and other substances catalyzed by flavins, but not the oxidation cata- lyzed by other dyes (11). Inhibition of the oxidations by monuron was however monitored as a reduction in oxygen consumption and was found to be more pronounced at low oxygen concentrations. Interaction of monuron with 02" produced from the flavins may be indicated by these results. Such an interaction would most likely convert 02" back to molecular oxygen and would appear as a net reduction in oxygen consumption. Interaction of monuron and diuron with 02" produced from photo- reduced flavins but not with 02" produced from other systems may sug- gest that the herbicides are not sensitive to 02", but they are specifically sensitized by flavins. Carbonyl-14C-labeled monuron reacted in the riboflavin/EDTA/light system. The radioactive products were partitioned between water and methylene chloride and analyzed by chromatography (Figs. 18 and 19). One major water-soluble product and several minor products soluble in either water or methylene chloride were 88 1.11: Unidentified Products in) In? Unchanged Nkuuuon n[ 15 - '9 S! x EE1°'_' c: 5 r- I ll 0 - -:- - 4- - I l .I I I l i l I l I I II I'TTI O 1 2 34 5 5 7 8 910111213141516 DISTANCE FROM ORIGIN (cm) Fig. 18. Radioactive products from the monuron/02" interaction and unchanged monuron recovered in the methylene chloride phase. Reaction mixture containing carbonyl-1”C-monuron, riboflavin, and EDTA was illum- inated and then extracted with methylene chloride. The methylene chloride phase was concentrated and spotted on silica-gel TLC plates. The radio- chromatogram was obtained from a developed plate by scraping off the gel in l-cm strips and measuring radioactivity. See "Materials and Methods’ for more details. 89 N 53 23 :3 e» I I I 1 b O T T dub-Ad N I one x 10" [mi 0 T (3 To .5 «a in I I I I I I, L 10 20 30 4O 50 FRACTION NUMBER 8 3 Fig. 19. Radioactive products from the monuron/02'- interaction recovered in the water phase. The water phase from the reaction mixture extracted with methylene chloride (Fig. 18) was concentrated and chroma- tographed on a Sephadex G-10 column (1.5 x 60 cm). Fractions of 4.0 ml were collected and assayed for radioactivity. 90 obtained. No liberation of 14CO2 could be detected during the course of the reaction, which indicates that hydrolysis of the amide bonds of monuron did not occur. Thus, non-radioactive products of monuron not detected by this procedure can be only methyl groups and/or chlorine. When the formation of the water-soluble products was completely preven- ted by using 500, the radioactivity corresponding to these products was recovered as unchanged monuron, and no other methylene chloride-soluble products were observed. This indicates that methylene chloride-soluble products are side-reaction products rather than photochemically produced intermediates from which the major water-soluble-product is formed by the subsequent action of 02 Therefore, these structural changes of the original monuron molecule do not appear likely to precede the monuron/02" interaction. Sweetser has reported that riboflavin or FMN converts monuron to an unidentified inactive product of high molecular weight (22). In the pre- sent study, illumination of monuron for 1 hr in presence of riboflavin alone resulted in 50% change of monuron to a methylene chloride-soluble product. This product was not formed when monuron was illuminated in presence of riboflavin and a reductant (methionine or EDTA). nor when the 02" was intercepted by $00. The flavin-monuron photoreaction des- cribed by Sweetser is not, thus. the precursor for the monuron/02" inter- action either. Identification of the major water-soluble product from monuron was attempted. The purified product was a yellowish solid with melting and boiling points above 350 C. Due to its high boiling point the mass spectrum of this compound could not be obtained. The UV spectrum resembled that of monuron and p-chlorophenylurea (E2, K, and B aromatic bands), 91 although the K-band was slightly shifted to a shorter wavelength and the B-band intensified. The UV spectrum indicated that riboflavin was not incorporated into the product. The compound was soluble in aqueous HCl, NaOH and HaHCO3 without undergoing hydrolysis. The infra red spectrum of the product was simpler than that of monuron and p-chlorophenylurea. The product was dechlorinated (no C-Cl band at around 1090 cm'l) and hydroxylated (C-O band at 1245 cm'1). Bathochromic shift (shift to the red) and intensification of the UV bands with changing the solvent from water to aqueous NaOH (pH 13.0) further indicated phenolic character of the product. The N-H stretching bands (around 3300 cm-1) resembled those of p-chlorophenylurea rather than of monuron, and thus the product may be demethylated. The product and monuron were compared for toxicity on 8-day-old seedlings of oats. Both chemicals in 10 0M solutions were applied to roots (nutrient solution) or to foliage (dipping twice). No toxicity symptoms or reduction of dry weight was observed with the product 10 days after application. Monuron reduced the dry weight by 61% when applied to the roots. Neither monuron nor the product reduced the dry weight of the seedlings when applied to the foliage. LITERATURE CITED 1. ASADA, K. AND K. KISO. 1973. The photooxidation of epinephrine by Spinach chloroplasts and its inhibition by superoxide dismutase: evidence for the formation of superoxide radicals in chlor0plasts. Agr. Biol. Chem. 37:453-454. 10. ll. 92 AUST, 5.0., D.L. ROERING, IAND T.C. PEDERSON. 1972. Evidence for superoxide generation by NADPH-cytochrome c reductase of rat liver microsomes. Biochem. Biophys. Res. Commun. 47:1133-1137. AUTOR. A.P. 1974. Reduction of paraquat toxicity by superoxide dismutase. Life Sci. 14:1309-1319. BABIOR, B.M., R.S. KIPNES. AND J.T. CURNUTTE. 1973. The production by leucocytes of superoxide: a potential bactericidal agent. J. Clin. Invest. 52:741-744. BEAUCHAMP, C.0. AND I. FRIDOVICH. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276-287. BLACK. C.C.. Jr. AND L. MYERS. 1966. Some biochemical aspects of the mechanisms of herbicidal activity. Weeds 14:331-338. FARRINGTON, J.A., M. EBERT, E.J. LAND, AND K. FLETCHER. 1973. Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides. Biochim. Biophys. Acta 314:372-381. FRIDOVICH, I. 1974. Superoxide dismutases. Adv. Enzymol. 41:35-97. GREGORY, E.M. AND I. FRIDOVICH. 1973. Oxygen toxicity and the superoxide dismutase. J. Bacteriol. 114:1193-1197. HALLIWELL, B. 1974. Superoxide dismutase, catalase and glutathione peroxidase: solutions to the problems of living with oxygen. New Phytol. 73:1075-1086. HOMANN. P. AND H. GAFFRON. 1963. Flavin sensitized photoreactions: effects of 3-(p-chlorophenyl)-l,l-dimethylurea. Science 141:905-907. 12. 13. 14. 15. 16. 17. 18. 19. 93 KOK, 8., H.J. RURAINSKI, AND 0.V.H. OWENS. I965. The reducing power generated in photoact I of photosynthesis. Biochim. Bi0phys. Acta 109:347-356. 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. 36:891-897. MC CORD, J.M. AND I. FRIDOVICH. 1969. Superoxide dismutase - an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244:6049-6055. MISRA. H.P. AND I. FRIDOVICH. 1971. The generation of superoxide radical during the autoxidation of ferredoxins. J. Biol. Chem. 246:6886-6890. MISRA. H.P. AND I. FRIDOVICH. 1972. The univalent reduction of oxygen by reduced flavins and quinones. J. Biol. Chem. 247:188-192. NISHIKIMI, M., N.A. RAO. AND K. YAGI. 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 46:849-854. PLIMMER, J.R., P.C. KEARNEY, AND U.I. KLINGBIEL. 1971. s-Triazine herbicide dealkylation by free-radical generating systems. J. Agr. Food Chem. 19:572-573. PREMA-KUMAR,|1., S.0. RAVINDRANATH, C.S. VAIDYANTHAN, AND N. APPAJI RAO. 1972. Mechanism of hydroxylation of aromatic compounds: II. Evidence for the involvement of superoxide anions in enzymatic hydroxylations. Biochem. Biophys. Res. Commun. 49:1422-1426. 20. 21. 22. 23. 24. 94 PREMA, K. AND K.P. GOPINATHAN. 1974. Involvement of the super- oxide anion in sulfoxidation. Biochem. J. 137:119-121. STANCLIFFE, T.C. AND A. PIRIE. 1971. The production of superoxide radicals in reactions of the herbicide diquat. FEBS Lett. 17:297-299. SWEETSER. P.B. 1963. Photoinactivation of monuron, 3-(p-chlorophenyl)- 1.1-dimethylurea, by riboflavin 5'-phosphate. Biochim. Biophys. Acta 66:78-85. WHITE, J.R., 1.0. VAUGHAN. AND W.-S. YEH. 1971. Superoxide radical in the mechanism of action of streptonigrin. Federation Proc. 30:1145 (Abstr.) ZWEIG, G., N. SHAVIT, AND M. AVRON. 1965. Diquat (l,l'-ethylene- 2,2'-dipyridylium dibromide) in photoreactions of isolated chloro- plasts. Biochim. Biophys. Acta 109:332-346. SUWIARY AND CONCLUSIONS 95 SUMMARY AND CONCLUSIONS The photochemical assay system for S00 generating 02" from riboflavin/methionine upon illumination and reducing NBT to blue formazan is reliable for determination of the enzyme in crude extracts from plant tissues. The only impurity in the crude extracts which may interfere with this assay is peroxidase. Peroxidase, however, is heat- labile. in contrast to the heat-stable SOD, and thus the interference can be avoided by heating the crude extracts before assaying. One unit of SOD has been defined as the amount that causes 50% inhibition of the N81 reduction, and thus far the enzyme has been quantitated on basis of % inhibition. The kinetics of the assay reaction indicated that % inhibition and enzyme units are not linear. More accurate determination of the enzyme can be made from the ratio of NBT reduction in absence versus presence of SOD, and defining 1 unit as the amount of enzyme for which this ratio equals 2. An equation for calculating the enzyme units from this ratio was derived from the kinetics 0f the reaction and was confirmed with a variety of crude extracts. Large quantities of SOD were found in shoots, roots, and seeds of corn, oats. and peas. On a dry weight basis shoots contain more enzyme than roots and both shoots and roots more enzyme than seeds. In seeds, the enzyme is present in both the embryo and the storage tissue. In lO-day-old seedlings, SOD accounts for 0.9 to 3.1% of the water-soluble protein. Quantitative differences exist between species and between 96 organs within a species. Furthermore, development of the SOD activity during seedling growth follows a different pattern in oats than in peas. The enzyme activity increases with age in oats, but not in peas. Con- versely, the activity increases with greening in peas, but not in oats. The extractable $00 of the plant Species studied is a copper-zinc enzyme. Manganese-enzyme accounting for less than 5% of the activity may be present in corn. A family of isozymes of the copper-zinc $00 was observed. The isozymic pattern varied with species. Variations in the isozymic pattern were also observed between organs within a species and were explained by the organelle specificity of the isozymes. The occurrence of SOD in plants indicates that 02" is formed in plant tissues. Variations in quantity and in isozymic pattern of SOD with species. organ. and stage of seedling growth may reflect Similar variations in the formation of the radical. Possible involvement of the 02" in herbicidal action and metabol- ism was studied by in vitro experiments. The ability of herbicides to either produce 02" or to react with 02" was examined by using model systems. Paraquat was reduced photochemically or enzymatically and produced 02’- upon reoxidation. Monuron and diuron were converted to non-toxic water-soluble compounds by photochemically produced 02". Formation of 02" within plants as a result of herbicide treatment, if it is in excess of the concentration which can be scavenged by the endogenous S00, may be related to the toxicity of the herbicides. Inter- action of herbicides with 02" in plants, on the other hand, may be a mechanism of herbicide metabolism. Much more research is needed, however. before conclusions can be made. "IIIIIIIIIIIIIIIII