Archives of Insect Biochemistry and Physiology 3:31-43 (1986) Superoxide Dismutase in the Housefly, Musca dornestica (L.) Thomas G . Bird, Marvin L. Salin, JohnA. Boyle, and James R. Heitz Department of Biockemisty, Mississippi State University, Mississippi State Four superoxide dismutase (SOD) (E.C. 126.96.36.199) isozymes were present i n whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg t o adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of t w o equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able t o protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed. Key words: superoxide dismutase, housefly, Musca domestica, enzyme purification and characterization, rose bengal, singlet oxygen, enzyme deactivation Acknowledgments: We thank Mr. William E. Poe, Department of Biochemistry, and Mr. Michael Aide, Department of Agronomy, Mississippi State University, for able assistance with the amino acid analysis and atomic absorption spectrophotometry, respectively. We also thank Dr. Paul A. Hedin, USDA-ARS, Boll Weevil Research Laboratory, Mississippi State, Dr. S.B. Ramaswamy, Department of Entomology, and Dr. Robert B. Koch, Department of Biochemistry, Mississippi State, for critical review of the manuscript. This work was supported by funds from the Mississippi Agricultural and Forestry Experiment Station MAFES, publication No. 5879. Received May 26,1985; accepted June 12,1985. T.C.B.’s present address i s Center for Alluvial Plains Studies, Delta State University, Cleveland, MS 38732. Address reprint requests to James R. Heitz, Department of Biochemistry, Drawer BB, Mississippi State, MS 39762. @ 1986 Alan R. Liss, Inc. 32 Bird et al INTRODUCTION As a consequence of living in an atmosphere enriched in oxygen, aerobic organisms have undergone a pressure to evolve enzymes capable of detoxifying deleterious by-products of oxygen metabolism. Among such enzymes are the class of metalloproteins known as superoxide dismutases.These enzymes detoxify the 02-by catalyzing a disproportionation reaction between two 02-molecules: Several isozymes of SOD* have been characterized with differences in amino acid sequence and metal content. CuZnSOD has been found in most eucaryotic organisms and consists of a dimer with a molecular weight of 32,000 and has 2 Cu and 2 Zn atoms per molecule [l-51. MnSOD has been found in bacteria as well as in the mitochondria1 matrix of plants and animals. The enzyme has a molecular weight of 40,000-90,000 depending on source and a variable metal content of 1to 4 atoms Mn per molecule. A third class, FeSOD, has generally been found in procaryotes [l],though exceptions have been reported [2,3].This isozyme has a molecular weight of about 40,000, consists of two subunits, and has a metal content of 1to 2 atoms of Fe per molecule. SODs from insects had been largely ignored until Lee et a1  purified and characterized CuZnSOD from the fruit fly, Drosopkila rnelunoguster. As a class of enzymes, the SODs are of particular interest to us owing to their postulated ability of interacting with '02and inhibiting its reaction [6,7] or by acting as protectant against the effects of ionizing radiation . Specifically, xanthene dyes, known to generate ' 0 2 as part of their photooxidative reaction, are now in use as insecticides . If SOD does afford a degree of protection to the organism that has been exposed to '02, a possible source of resistance to the pesticide may exist. Therefore, the intent of the current study is to purify and characterize CuZnSOD from housefly prior to initiating a study on '02and its effect on housefly SOD. We also report results of in vitro studies into the interaction of '02and CuZnSOD from house fly. MATERIALS AND METHODS Newly emerged, adult houseflies from a colony initially established in 1977 with wild insects from a caged-layer poultry house in Pelahatchie, MS and maintained in the Department of Biochemistry, Mississippi State University, were used in the purification studies. Other stages used are noted with each study. The adult fly rearing colony was maintained on dry milk, sugar, and *Abbreviations: CM-Sepharose = carboxyrnethyl Sepharose; DEAE-Sephadex = diethylaminoethyl Sephadex; pl = isoelectric point; LDH = lactic dehydrogenase; M, = relative molecular weight; M W = molecular weight; PAGE = polyacrylamide gel electrophoresis; RE3 = rose bengal; SDS-PACE = sodium dodecyl sulfate PAGE; lo2 = singlet oxygen; 0 2 -= superoxide anion; SOD = superoxide dismutase. Housefly Superoxide Dismutase 33 H20 with a 14L:lOD photoperiod at approximately 27°C. The standard larval diet used throughout contained 7 ml of malt solution (50 ml of commercial malt extract plus 100 ml H20), 27 ml of a solution of brewer’s yeast (66 g of yeast in 540 ml H2O; ICN Nutritional Biochemicals; Cleveland, OH), and 190 ml H201100 g of wheat bran. The mixture was allowed to ferment 24 h prior to introduction of eggs or larvae. Gravid females laid their eggs directly onto the larval media. The eggs were allowed to develop, and first-instar larvae were transferred to fresh media. CuZnSOD from 1kg of houseflies was purified according to the procedure of McCord and Fridovich  with two additional steps. The presence of contaminants not resolved by the original procedure led to the inclusion of a CM-Sepharose column following the usual DEAE-Sephadex chromatography. The sample was loaded onto the 2.5 x 20 cm CM-Sepharose column in 5 mM, pH 6.0, phosphate buffer and eluted without adhering to the matrix. Following gel filtration on Sephadex G-75 ultrafine, the presence of impurities necessitated development of a method for preparative PAGE. A Canalco PREP-DISC@unit, outfitted with a I’D-150 gel column, was employed for preparative PAGE. The analytical electrophoresis method described below was modified by increasing the polyacrylamide gel concentration to 15% in the resolving gel. A 1.5-cm resolving gel and 3.0-cm concentrating gel were supported by fine mesh nylon netting across the column end. Flow rate of the elution buffer was controlled by a Gilson Minipuls 2 peristalic pump at 0.5 mllmin, and temperature was maintained at 4°C by circulating 50% ethylene glycol through a refrigeration unit. A driving force of 4 mA was applied until the sample entered the resolving gel and was then increased to 8 mA for the duration of the run. The unit of SOD activity was initially defined by McCord and Fridovich  in an indirect assay. Xanthine oxidase is added in sufficient quantity to a reaction mixture containing xanthine (5 x lo-’ M) as an oxidizable substrate and ferricytochrome c (1 x lo-’ M) as a reducible substrate in phosphate buffer (pH 7.8, 50 mM). A rate of reduction measured at 550 nm of 0.025 absorbance unitslmin at 22.5 1°C is desired. Under these conditions, McCord and Fridovich defined 1 unit of enzyme activity as the amount of SOD required to inhibit the rate of ferricytochrome c reduction by 50% (0.0125 absorbance unitslmin). A drawback to expressing the unit of activity in this manner is the lack of linearity over a wide range of SOD concentrations. By using the equation of Asada et a1 : (Vlv) = 1 + K’ [SOD] where V and v represent the reaction rate of the assay in the absence and presence of SOD, Giannopolitis and Ries [lo] demonstrated linearity over a wider range of SOD concentrations when the reaction rate constant, K’, is set equal to 1.By derivation they arrived at the following expression: SOD unitslml = [(Vlv) - 11 (dilution factor) It is this expression of enzyme activity that the present paper uses. 34 Bird et al Protein levels were estimated by either the modified Lowry method of Schacterlee and Pollack [ll]or the differential absorbance method of Murphy and Kies 1121 as indicated. The use of two methods for protein assay was necessitated by numerous nonproteinaceous compounds which absorb at 215 nm and interfered with the latter method in the early stages of purification, thereby giving highly erroneous results, and also by rapidly diminishing enzyme levels in the latter stages of purification which made use of the destructive Lowry method infeasible. Electrophoresis on 7.5% polyacrylamide gels was performed according to the procedures of Davis  and Ornstein 1141 and stained by the method of Beauchamp and Fridovich . CuZnSOD isozymes were identified by convention on polyacrylamide gels as bands sensitive to 1 mM KCN , whereas bands identified as MnSOD were insensitive to both KCN and 1 mM H202 m1. Development of SOD isozymes from egg to adult was followed by first collecting a large number of freshly laid eggs. A representative sample was frozen at -70°C until needed for the comparative study, but the remainder were allowed to develop. Frozen samples did not differ from fresh ones when compared electrophoretically. Larvae or pupae were taken every 24 h until adult emergence and frozen. Upon adult emergence, accumulated samples were prepared for electrophoresis. Distribution of the isozymes in the body tagma (head, abdomen, and thorax) was determined by sectioning live insects, freezing immediately in liquid N2, and electrophoresing as described above. Molecular weights were measured by both gel filtration chromatography and SDS-PAGE. For the former, a Sephadex G-200 column (40-120 mesh, 1.5 x 70 cm) was equilibrated with standard phosphate buffer (50 mM, pH 7.8) and calibrated with ribonuclease A (MW 13,700), chymotrypsinogen A (MW 25,000), 6-lactoglobulin (MW 36,800), ovalbumin (MW 45,000), bovine serum albumin (MW 66,000), and aldolase (MW 158,000, Sigma, St. Louis). SDSPAGE was performed by the method of Laemmli 1171. Standards used to calibrate included egg white lysozyme (MW 14,300), 6-lactoglobulin (MW 36,800, subunits of 18,400), porcine stomach mucosa pepsin (MW 34,700), ovalbumin (MW 45,000), and bovine serum albumin (MW 66,000). Copper and zinc levels were determined on a Perkin-Elmer model 305B atomic absorption spectrophotometer. Purified enzyme was dialyzed extensively against 0.1 mM ethylenediamine tetraacetic acid in deionized water prior to the determination. The amino acid composition of the three CuZnSOD isozymes in one solution was determined after hydrolysis in vacuo in 6 M HC1 at 110°C for 24 h followed by analysis on a Beckman model 120C amino acid analyzer. Tryptophan was measured spectrophotometrically using the N-bromosuccinimide method of Spande and Witkop 1181. The PI of the housefly CuZnSOD was determined by the method of Salin and Bridges 1191 using a DesagalBrinkman double-chamber gel isoelectric focusing apparatus. The absorbance spectrum was determined on a PerkinElmer model 552 spectrophotometer. Purified, adult housefly CuZnSOD was used in two in vitro experiments to determine the effects of RB-generated Housefly Superoxide Dismutase 35 on the enzyme. In the first RB (1mM) and CuZnSOD were prepared in 1 ml phosphate buffer (50 mM, pH 7.8). The amount of CuZnSOD present was sufficient to halve the reduction rate of cytochrome cox to cytochrome C,,d when a 10-p1aliquot was used in the previously described enzyme assay. After 30 min dark preincubation the test tube containing the solution was placed into a cooling jacket constructed of a chromatography jar and a recirculating water bath to maintain a constant temperature of 25°C for the exposure period. The solution was then illuminated with a 650-W Sylvania sun gun. Activity was measured at 3-min intervals for 60 min. In the second experiment housefly CuZnSOD was tested as a protectant for a second enzyme, LDH, against the effects of lo2.Five different assay tubes were prepared for each of the four replicates: a dark control with LDH only, a dark control with LDH and RB, an illuminated tube with LDH only, an illuminated tube with LDH and RB, and an illuminated tube with LDH, RB, and 3 pM housefly CuZnSOD. Tubes were maintained as before at 25°C. Each tube contained 100 pl of bovine heart LDH (550 units; 1 unit converts 1 pM pyruvate to L-lactate per min at pH 7.5, 37°C) in 15 ml phosphate buffer (50 mM, pH 7.5). RB was added to produce absorbance = 0.80 at 556 nm, which previously was shown to produce an acceptable rate of LDH inactivation when illuminated. LDH activity was measured at 2-min intervals using previously published methods . An aliquot of the illuminated LDH-RB mixture was added to an enzyme assay mixture containing NADH (0.2 mM) and pyruvate (0.5 mM) in phosphate buffer (50 mM, pH 7.5) at 37°C. The conversion of NADH to NAD+ was measured at 340 nm until greater than 80% LDH deactivation was observed. ' 0 2 RESULTS Electrophoresis and activity staining of various samples without inhibitors always showed four achromatic bands. All four resolvable bands were present in each of the adult body tagma (Fig. l A , B, and C) and from egg to adult (data not shown). In the presence of the inhibitors KCN or H202, the three fastest migrating bands (bands 2, 3, and 4) were eliminated but the slowest (band 1)was not affected (Fig. 1D and B). Based on the known sensitivities of the various SOD isozymes [15, 161, it was concluded that the triplet peaks were CuZnSOD isozymes and that the slowest band was MnSOD. There was no evidence for FeSOD, in keeping with the general observation that this isozyme is found predominantly in prokaryotes [l]. The crude homogenate had a specific activity of 7 units Img protein (Table 1).Treatment of the sample with organic solvents eliminated a large portion of the contaminant while not affecting the total activity. Chromatography over three columns resulted in a 132-fold increase in specific activity while only half of the total activity was lost. Contaminating proteins were effectively eliminated by preparative electrophoresis, with the faster-migrating contaminants eluting prior to most SOD activity. Other contaminants were unable to enter the 15% PAGE resolving gel and were visible as a dark-brown band at the concentrating ge1:resolving gel interface. The inclusion of this step increased the specific activity approximately 2.9-fold over the Sephadex 36 x,- Bird et al D W 0 t 0 I ? 0 In n a c 2cm Fig. 1. SOD activity from newly emerged, adult houseflies localized o n polyacrylamide electrophoresis gels. Results from the body tagma (head, A; thorax, B; and abdomen, C) are represented by enzyme stained without inhibitor present. Whole-body homogenates were electrophoresed and stained in the presence of KCN (D) or H 2 0 2 (E) after the method of Beauchamp and Fridovich [I51 and Asada et al [I61 to determine the presence of MnSOD, peak 1; CuZnSOD, peaks 2 , 3 , and 4; and/or FeSOD. TABLE 1. Purification of CuZnSOD From Newly Emerged, Adult Houseflies Crude homogenate Acetone precipitate DEAE-Sephadex CM-Sepharose G-75 Sephadex Preparative electrophoresis Specific activity (unitsimg protein) Volume (mu Total protein (mg) Total units 7,075 57,770a 382,100 7 100 1 690 3,367a 340,900 101 89 15 1,080 1,090 275 410 1,159a 672a 223b 56b 315,400 272,500 195,800 141,040 272 406 878 2,519 83 71 51 37 41 61 132 381 'Protein measured by method of Schacterlee and Pollack [ll]. %otein measured by method of Murphy and Kies . Percent recovery Fold purification Housefly Superoxide Dismutase 37 G-75 step. Analytical electrophoresis indicated that the purified sample contained three CuZnSOD isozymes but no other bands, whereas only a single peak of SOD activity was observed when chromatographed on Sephadex G200. Analysis of PAGE gels according to the method of Hedrick and Smith  indicated that the purified isozymes were charge isomers (data not shown). An M, for these CuZnSOD isozymes as measured by Sephadex G-200 gel filtration was approximately 34,800. The determination of M, by SDS-PAGE  from a solution containing all of the housefly CuZnSOD isozymes produced a single band corresponding to approximately 16,000 when heated at 100°C for 5 min in both the presence and absence of 2-mercaptoethanol. The UV-absorbance spectrum of a solution containing all three of the housefly CuZnSOD isozymes (Fig. 2) was essentially the same as that of CuZnSOD from other sources. Absorption shoulders were observed at 264 and 259 nm. Extinction coefficients corresponding to these peaks were 7,850 M-' cm-' and 7,890 M-' cm-' based on an M, of 32,000. Sufficient enzyme was not available to generate the spectrum in the visible region. Analysis for metal content of the enzyme showed the presence of 1.6 mol of copper and 1.7 mol of zinc per mol of dimer. The amino acid composition of the three CuZnSOD isozymes based on a dimeric molecular weight of 32,000 is shown in Table 2 along with that of several other organisms. The most noteworthy features of the housefly CUZIISOD amino acid profile are the absence of tryptophan and the pres- WAVELENGTH, nm Fig. 2. Absorbance spectrum of housefly CuZnSOD from newly emerged adults in the U V region. A protein concentration of 0.6 m g h l was used to generate this spectrum. An M, of 32,000 was used in calculations. All three isozymes were present in solution. 38 Bird et al TABLE 2. Comparison of the Amino Acid Composition of CuZnSOD From Musca dornestica and Several Other Sources Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine leucine Tryptophan Phenylalanine Lysine Histidine Arginine Tyrosine Total residues Chicken livera Spinachb 32 18 14 23 12 50 22 14 28 4 14 16 0 8 20 14 8 2 299 35 28 10 20 17 42 23 4 28 2 6 22 0 6 13 14 7 0 277 Ponyfish' 38 20 19 28 12 46 33 - 20 5 13 21 2 11 22 13 6 3 312 Fruit flf 30 15 15 18 10 44 20 8 26 2 14 12 - 10 18 14 6 2 264 House- flye 40 16 25 27 17 38 27 18 10 5 9 19 0 11 21 10 9 4 306 aWeisiger and Fridovich . bAsada et a1 . 'Martin and Fridovich . dLee et a1 141. eDetermination of residues made on a solution containing three housefly CuZnSOD isozymes. ence of four tyrosine residues per dimer. The 18 cysteine and five methionine residues exceed the numbers typically observed in CuZnSOD, whereas the 10 histidines and 10 valines were fewer than typically observed. A PI of 4.85.1 was measured from the matrix of the isoelectric focusing gel and reflects a protein containing a high percentage of aspartic acid and glutamic acid residues. Housefly CuZnSOD, incubated in the dark for 30 min and illuminated for 60 min, was not affected by RB generated ' 0 2 (Table 3). This lack of effect by *02 was not surprising considering that the substrate for SOD is the high energy oxygen radical, 02-. LDH lost no activity in the presence or absence of RB when not illuminated (data not shown) but was rapidly deactivated when illuminated (Table 4).At 3 pM housefly CuZnSOD provided no protection for LDH, though this level of SOD is sufficient to almost completely inhibit the conversion of cytochrome cox to cytochrome c,,d in the bioassay. To ensure that the concentration of housefly CuZnSOD was not too low to provide protection, 15 pM additional bovine heart CuZnSOD was added to the reaction vessel with no observable effect. DISCUSSION The purification procedure used in this study was more complex than the original isolation of CuZnSOD from bovine erythrocytes by McCord and Housefly Superoxide Dismutase 39 TABLE 3. Effects of Rose Bengal on Purified Housefly CuZnSOD From Newly Emerged Adults In Vitro Timea (min) Activityb (unitsiml f SD) 0 15 30 45 60 25.8 26.2 26.8 26.2 25.8 + 1.6 f 0.7 f 1.8 f 0.9 i 0.9 aTest solution containing rose bengal (1 mM) and housefly CuZnSOD (all 3 isozymes present) was preincubated in the dark for 30 min and then exposed to a 650-W light source for 60 min. bActivity measured using method of Asada et a1 19,101. See text for details. Each value represents triplicate determinations. TABLE 4. Effect of Rose Bengal on Lactic Dehydrogenase in the Presence and Absence of Purified Housefly CuZnSOD* Time (min) 0 2 4 6 8 10 12 % Inhibition Controla 0 7.1 f 2.9 32.1 8.3 46.6 6.3 60.8 f 7.1 75.0 k 6.7 84.1 + 8.1 * * + SD Plus CuZnSODb 0 7.3 f 2.1 29.6 + 2.2 49.8 3.3 61.3 f 1.0 73.8 f 2.1 80.5 + 4.9 *Purified housefly CuZnSOD from newly emerged adults was actually a solution of three electromorphs (see text). "Test solution containing RB (Abs = 0.8 at 556 nm) and LDH (550 units) in 15 ml phosphate buffer (50 mM, pH 7.5) illuminated with a 650-W light. Assay after Callaham et a1 1201. milliliter of solution from "a" plus 3 pM (96 p g ) housefly CuZnSOD. See text for details. Fridovich . Contaminants were observed at the final gel filtration step when the unmodified method was used, and as a result, cation exchange chromatography on CM-Sepharose and preparative electrophoresis were incorporated. M . domestim CuZnSOD proved to be highly stable to salting out with dibasic potassium phosphate in the presence of chloroform-ethanol, whereas D. melanogaster enzyme quickly lost activity at this step . The level of purification and specific activity using DEAE-Sephadex was lower in this study than that in studies using erythrocyte sources. Typically, 60- to 300fold purification is observed at this step [5,22-241; however, only a 41-fold purification was measured here. Sephadex gel filtration gave a 132-fold purification with a specific activity of approximately 880. Gel filtration is the last step in many CuZnSOD purifications, and specific activities approaching 2,600 were frequently observed; however, inclusion of preparative electrophoresis was required to attain a comparable level of purity [2,5,24]. Lee et 40 Bird et al a1 141 reported a much higher specific activity for D. melanoguster of approximately 4,800. In parallel studies with the bovine enzyme, D. lnelunogasfer CuZnSOD was found to be 1.6 times more active. This high activity in the fruit fly led them to conclude that the high level of resistance to ionizing radiation observed in Drosophilu is due to this unusually active CuZnSOD. SOD activity in the Lee et a1  study was measured by the method of McCord and Fridovich , whereas in this study it was expressed in the form of Asada units . Therefore, caution should be taken when comparing the results of these two studies because of difference in the expression of assay results. The aggregation phenomenon reported by Salin and Wilson  for the porcine enzyme was also observed in the housefly protein. Enzyme purified by gel filtration and homogeneous with respect to molecular weight at approximately 34,800 was stored at 4°C. Subsequent chromatography of the fraction on Sephadex G-200 revealed the presence of a high-molecularweight, 254-nm absorbing peak that possessed no SOD activity and consistently eluted in the void volume. It was assumed that this high-molecularweight fraction was aggregate CuZnSOD, which could be seen as a bluegreen precipitate on standing in purified sample. Protein measurement of a filtered sample of this purified CuZnSOD sample indicated that protein loss had occurred while the specific activity was unchanged. Salin and Wilson 131 suggested that the self-association was due to interchain sulfhydryl interactions and could be prevented by addition of a thiol reagent. We also observed that 2-mercaptoethanol or dithiothreitol prevented formation of the precipitate, though it was not possible to reverse the process. MnSOD and CuZnSOD were both found in housefly tissues. It is now known that MnSOD is a mitochondria1enzyme and that CuZnSOD is located in the cytosol, though exceptions have been noted 113. Three CuZnSOD isozymes were identified at all ages and in each of the body tagma along with a single MnSOD. These results indicated that all isozymes are present in each of the body divisions. No attempt was made to determine the presence of the isozymes in individual organs either quantitatively or qualitatively. The relationship of the CuZnSOD isozymes to one another has been investigated previously [14,24-281. Multiple isozymes have been reported but not characterized in other insect species. Lorimer  observed three isozymes in the forest tent caterpillar, Mulucossomu disstriu, with as many as four in some individuals, and Bartlett 1301 reported two isozymes in the pink bollworm, Pectinorphoru gossypielh. Neither study specified the forms as CuZnSOD or MnSOD, but it is likely that the most common isozymes observed were analogous to the CuZnSOD electromorphs and MnSOD of this study. Lee et a1  reported only one CuZnSOD and one MnSOD in D. melanoguster. The amino acid profile of housefly CuZnSOD is presented in Table 2 along with those of several other species. It is obvious that the housefly enzyme contained a greater number of aspartic acid, serine, proline, and cysteine residues but fewer valine residues. Four tyrosines were present per dimer of housefly CuZnSOD, whereas only two per dimer are generally reported. Housefly Superoxide Dismutase 41 Drosophilu and chicken liver are representative of those enzymes. Martin and Fridovich [27l examined seven marine fish species and typically found 3-5 tyrosine residues in each of these. Tryptophan was absent from Muscu and all other species in Table 2 except ponyfish. The absence of the tryptophan in these species including housefly is reflected by the UV-absorbance maximum centered at 258 nm while ponyfish has a higher maximum at 265 nm (Table 5). The extinction coefficient of housefly CuZnSOD is only onehalf the value of fruit fly enzyme but is in the same range as that in spinach and chicken liver. The 1.6 mol of copper and 1.7 mol of zinc per mol of enzyme dimer are in close agreement with the values reported for the other species. Determination of subunit size by SDS-PAGE led to the conclusion that each molecule of active enzyme is composed of two associated subunits of approximately 16,000. Based on this, the molecular weight of the dimer would be in the 32,000 range rather than 34,800 measured by gel filtration. M . domesticu CuZnSOD is not significantly different from CuZnSODs isolated from other organisms. Indeed the similarities are striking with respect to amino acid composition, molecular weight, metal prosthetic groups, PI, and ultraviolet absorbance spectrum as shown in Tables 2 and 3. The PI is the most dissimilar value, reflecting the variation in acidic amino acid residues. The housefly CuZnSOD data presented here actually represent a heterogeneous mixture of at least three resolvable isozymes. Rose bengal is one of a large group of dye compounds known to generate *02 in illuminated aqueous solutions. lo2generated in this manner is responsible for many biological effects including reactions with amino acids, nucleic acids, lipids, tocopherols, polysaccharides, and various enzymes . Housefly CuZnSOD, however, was found to be unaffected when exposed to '02for a period of 1h. Forman et a1  investigated bovine CuZnSOD and found it was also unaffected by lo2when its full metal complement was present. Removal of the metals led to a rapid, irreversible deactivation of the apoenzyme by the destruction of 3.6 histidine residues per molecule. Presumably, photooxidizable amino acid residues are not accessible to ' 0 2 as a TABLE 5. Comuarison of CuZnSOD From Musca domesticu and Several Other Sources Chicken livera Spinach' Ponyfish' Molecular weight Metal content per dimer Copper Zinc UV-absorbance peaks (nm) Extinction coefficient M-cm-* 30,400 32,200 32,000 8,750 9,920 - VI 5.35-6.75 - 7.8-8.2 1.8 1.6 258 2.2 2.2 258 - 265 'Weisiger and Fridovich 1311. 'Asada et a1 . 'Martin and Fridovich . dLee et a1 . eValues measured from a solution of three CuZnSOD isozymes Fruit flyd 32,000 2.1 2.2 258 13,200 5.3 Houseflye 32,000 1.6 1.7 258.9 7,890 4.8-5.1 42 Bird et at result of evolutionary pressures to eliminate residues that react with highenergy oxygen. Studies on the relationship of lo2and 02-have led at least two groups [6,nto suggest that SOD is capable of interacting with lo2and inhibiting its reaction. This was not the case in this study, since LDH exposed to lo2was rapidly inactivated when 3 pM housefly CuZnSOD was present or absent. The possibility of too low a CuZnSOD level was also precluded by addition of 15 pM bovine CuZnSOD. The results were identical, indicating that either 02-was not involved in the deactivation of LDH, though its generation by RB could not be ruled out, or housefly CuZnSOD was unable to detoxify the lo2molecule. Use of the xanthene dyes as pesticides presents another mechanism for toxicological study. In two in vitro tests we saw no indication that housefly CuZnSOD affords protection to the insect against an oxygen species (lo2) related to its primary substrate (02-); however, the possibility exists that the in vivo situation is different. 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