Superoxide Dismutase Activity in Bovine Milk Serum

867

M. KORYCKA-DAHLl, T. RICHARDSON I• and C. L. HICKS2

Department ofFood Science, University of Wisconsin, Madison, Wisconsin 53706, and Department ofAnimal Sciences, University ofKentucky. Lexington. Kentucky 40506

(Received for publication January 22,1978)

ABSTRACT have been implicated in lipid peroxidation and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/11/867/1649583/0362-028x-42_11_867.pdf by guest on 02 October 2021 Superoxide dismutase activity was shown to be present in bovine milk destruction of biological membranes (8,15.17,20,30), as serum and was quantifted by measuring the capacity of retentate from well as degradation of macromolecules (21). The SOD dialyzed milk serum to inhibit reduction of cytochrome c by which catalytically destroys superoxide anion is thought xanthine-xanthine oxidase-generated superoxide anion. One unit of enzyme was defined as the quantity of superoxide dismutase which to protect living cells from oxidative damage (7,15,17). inhibits cytochrome c reduction by 20o/o. By this definition 19,500 units Presumably, any biological system in which oxygen is of enzyme were present per liter of retentate from dialyzed milk serum. present and superoxide anion is normally produced is This amount is equivalent to about 2.4 mg of purified bovine likely to contain SOD as a defense against oxidative er)1hrocyte superoxide dismutase per liter. Polyacrylamide gel reactions initiated by oxygen species (24). Bovine milk electrophoresis of a partially-purified superoxide dismutase from acid whey. followed by staining for enzymic activity, confirmed the presence has several systems present which are able to generate of the enzyme in milk serum which was identical in electrophoretic superoxide anion. Among these are enzymes such as properties to those of bovine er)1hrocyte copper-zinc superoxide xanthine oxidase (7,14) and lactoperoxidase (11.14). The dismutase. Pasteurization at 63 C for 30 min did not decrease spontaneous oxidation of tetrahydropteridines (27), superoxide dismutase activity in milk serum. Heating of purified bovine (26), (25) erythrocyte-superoxide dismutase at 100 C for 1 min resulted in almost hemoglobin reduced flavins and quinones complete loss of enzymic activity. whereas the partially-purified results in formation of superoxide anion, and these can superoxide dismutase from acid whey still retained 40o/oofthe original be in milk. Therefore, the possibility that SOD exists in activity under these conditions. Bovine milk superoxide dismutase may the secretory cell, and secondarily in milk, was be an important naturally-occurring antioxidant for increasing investigated. oxidative stability of milk and other dairy products. In this study we confirm the occurrence of SOD activity in serum of bovine milk, as previously reported The enzymic activity of a blue-green metalloprotein (9); however, the quantity of SOD present in milk from present in bovine erythrocytes, known also as erythro our source was approximately 10-fold greater than that cuprein, was discovered by McCord and Fridovich in reported by other authors (1,10.12). A simple procedure 1969 (22.23). The protein was subsequently renamed for isolation of partially-purified SOD from bovine milk superoxide dismutase (SOD) because it catalyzes the has been elaborated and electrophoretic mobility and dismutation of superoxide anion (OT) radical to thermostability of the enzyme obtained from this hydrogen peroxide and triplet oxygen: procedure have been examined. The importance of SOD for the oxidative stability of milk is discussed.

MATERIALS AND METHODS Since that time, SOD has been observed widely in Enzymes nature. It has been isolated and characterized from many Enzymes were purchased from the following sources: milk xanthine animal, plant and microbial sources (7,19,28). At the oxidase from ICN Pharmaceuticals, Inc., Cleveland. OH; beef liver time this work was completed (9), Hill (1 0) and later catalase from Boehringer Mannheim Biochemicals. Indianapolis, IN; Asada (1) reported the occurrence of SOD in bovine purified bovine erythrocyte superoxide dismutase from Truett milk. More recently, Holbrook and Hicks (12) reported Laboratories, Dallas, TX. A partially-purit1ed bovine milk superoxide on the variation of SOD in bovine milk and measured dismutase was prepared from acid whey by fractionation with ammonium sulfate and ion exchange chromatography (Fig. 1). concentrations of enzyme similar to those found by Hill Reagents (10). Xanthine monosodium salt was purchased from ICN Pharmaceut The production of superoxide anion in many biological icals. Inc. Horse heart muscle ferricytochrome c and ethylenedi processes is well documented (4, 7).It is generally thought aminetetraacetic acid disodium salt (EDT A) were from Aldrich (7,20) that generation of this radical might lead to the Chemical Co., Milwaukee, WI. Bovine serum albumin (fraction V), formation of other oxidative species such as hydrogen L-methionine and nitro blue tetrazolium chloride (NBT) were from peroxide, singlet oxygen and hydroxyl radical, which Sigma Chemical Co., St. Louis, MO. The reagents. for polyacrylamide gel electrophoresis, carried out as described by Davis (5). were obtained 1 University of Wisconsin. from Eastman Organic Chemicals. Rochester. NY. All other chemicals 'University ofKentucky. were reagent grade. Deionized water was used in all assays. 868 KORYCKA-DAHL. RICHARDSON AND HICKS

rlsw akim milk z 6 ':;) @ 0: u UJ ~ (5 12 (J ~ ~ 201------fl ! +-- Salt fractionation 65-?0% aaturatior: witb (N.!:! ) bC l ! 4 2 4 iD I Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/11/867/1649583/0362-028x-42_11_867.pdf by guest on 02 October 2021 ..,..__ C€ntri.fug+: 15, ;

an increase in enzyme specific activity of about 260-fold, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/11/867/1649583/0362-028x-42_11_867.pdf by guest on 02 October 2021 indicating the enzyme is not associated with the casein fraction. SOD isolated from bovine milk according to procedures in Fig. 1, and commercial bovine erythrocyte Figure 3. Inhibition ofcytochrome c reduction by: curve l: 2.0 IJ.g/ml SOD were simultaneously subjected to polyacrylamide of bovine erythrocyte superoxide dismutase (SOD); curve 2: 25 !Jl of dialyzed milk sero.m retentate; curve 3:25 iJ[ of dialyzed milk sero.m gel electrophoresis. After staining for SOD activity, two retentate boiled for 10 min. Cytochrome c was complete(v reduced closely migrating achromatic bands located at the same using the xanthine-xanthine oxidase system (32). distance from the cathode were detected for the enzymes from both sources (Fig. 5). The observed electrophoretic ~ patterns of the enzymes were similar to those for the tJ i3 e ~ Ul w ~ 0 ~40 TWO ACHROMATIC BANDS tJ WITH SOD ~ I ACTIVITY ~ 0_'£' _____j_ ____j______, 0 .050 .100 .150 .200 DIALYZED MILK SERUM RETENTATE CmD Figure 4. Inhibition of cytochrome c reduction by dialyzed milk sero.m retentate. Superoxide anion was generated by xanthine-xanthine oxidase reaction. Each point on the curve represents an average of two measurements, each in duplicate. Bars represent upper and lower limits ofvalues. quantities (up to 0.1 ml) of retentate added, and, A B subsequently, this range was used for assaying of SOD Figure 5. Disc gel electrophoresis of superoxide dismutase. stained activity in serum. for enzyme activity with nitro blue tetrazolium in the presence of photogenerated superoxide anion. Gel A. 2 IJ.g purified bovine Concentration of SOD in bovine milk serum was erythrocyte superoxide dismutase. Gel B, 150 IJ.g ofprotein from erode calculated from the standard curve (Fig. 2) prepared for enzyme preparation isolated from bovine milk as shown in Fig. 1. TABLE 1. Superoxide dismutase activity in bovine milk sero.m assayed by inhibition ofcytochrome c reduction using the xanthine-xanthine oxidase

Proteina Superoxid01 dismutase Sample (mg/ml) ;;g/ml unitsb;ml unitsb/mgprotein

serum r nt:m."t" 8.07 0.18 2.44 :t: 1.20 19.5 :t: 9.6 2.4 :t: 1.2 Dialyzed serum retentate pasteurized at 63 C for30 mind 7.89 :t: 0.09 2.31 :t: 0.60 18.5 :t: 4.8 2.3 :t: 0.5 Crude preparation of bovine milk superoxide dismutased 0.48 :t: O.Ql 36.75 :t: 4.36 294.0 32.0 612.5 :t: 67.4 concentration was assayed by method of Lowry et al. U8). units were calculated according to the definition given in the Materials and Methods. cMean and SD calculated from eight separate samples. dMean and SD calculated from four separate samples. 870 KORYCKA-DAHL. RICHARDSON AND HICKS copper-zinc SOD from bovine erythrocytes reported by the SOD activity present in milk remains uncertain at the other authors (2.3.19). Therefore, polyacrylamide gel present time. The spontaneous development of oxidized electrophoresis further confirmed the presence of SOD flavor observed in certain fluid milks (29) might be in milk, and suggested that milk SOD is probably related to an insufficient amount of endogenous SOD in identical to the copper-zinc enzyme present in bovine milk. Studying the correlation between the amount of erythrocytes. There is a possibility that SOD derived endogenous SOD in milk and the susceptibility of milk to from bacterial or leukocytic cells is also present in milk. spontaneous oxidation might add valuable information However, those cells have manganese- and/or iron for explanation and prevention of these problems. containing SODs which migrate more toward the Recently, Holbrook and Hicks (12) concluded that the cathode during gel electrophoresis (7.28). No bands concentration of SOD in individual cow's milk did not representing those enzymes were detected on the account for the large variation in oxidative resistance in polyacrylamide gels. raw milk that had not been exposed to light. Purified SOD was less stable to heat than SOD in Thus the endogenous level of SOD may be insufficient Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/11/867/1649583/0362-028x-42_11_867.pdf by guest on 02 October 2021 crude preparations (Table 2). When the same amount of to act as an antioxidant under certain conditions. enzyme in either the pure or the crude form was heated Apparently milk does not have sufficient SOD to prevent at 100 C for 1 min, almost all activity of the highly oxidation and degradation of milk constituents by purified enzyme was abolished, whereas about 40o/o of photogenerated superoxide anion upon exposure of milk the initial activity was still present in the crude to light (16); however, the SOD may be adequate to preparations. Heating at 100 C for S min, however, scavenge any superoxide anion formed during storage in also destroyed enzymic activity in the crude preparation. the dark. Therefore, it is important to determine the Low-temperature pasteurization of milk serum (63 C, conditions which lead to formation of superoxide anion 30 min) did not decrease SOD activity to any great extent and other active oxygen species. (Table 1). About 96 o/o of the original SOD activity Additional research may indicate that oxidation remained after pasteurization. SOD is considered to be of milk and other food products could be slowed or one of the most stable enzymes with regard to prevented by addition of enzymes such as SOD to destroy temperature. pH changes, storage conditions and superoxide anion and catalase to destroy hydrogen resistance to many denaturating reagents (6,31). The peroxide, thus providing a new class offood antioxidants. greater resistance of the SOD enzyme to thermal inactivation in the presence of whey proteins should be ACKNOWLEDGMENTS beneficial for preserving SOD antioxidative activity in thermally processed dairy products. This research was supported by the College of Agricultural and Life Sciences. University of Wisconsin-Madison. and by a grant from the TABLE 2. Heat-inactivation ofsuperoxide dismutase (SOD) purified Cooperative State Research Service. United States Department of from bovine erythrocytes or crude superoxide dismutase fractionated Agriculture. from of bovine milk. a % Heating time (min) REFERENCES 1. Asada. K. 1976. Occurrence of superoxide dismutase in bovine 0 100.0 100.0 milk. Agr. Bioi. Chem.40: 1659-1660. 1 4.3 ± 0.2 40.4 ± 1.0 2. Beauchamp. C.. and I. Fridovich. 1971. Superoxide dismutase: 5 0 1.8 ± 0.2 lO 0 0 Improved assay and an assay applicable to acrylamide gels. AnaL Biochem. 44:276-287. containing 2.5 )lg/ml of erythrocyte or milk enzyme in 3.Bohnenkamp, W .. and U. Weser. 1975. Superoxide dismutase potassium phosphate buffer, pH 7.8, were incubated at 100 C. micro-assay in biological material. Hoppe-Seyler's Z. Phys. Chern. Aliquots were removed at the indicated time intervals, cooled in an ice bath. and assayed for SOD activity in terms of their inhibition of the 356:747-754. reduction of cytochrome c in the xanthine-xanthine oxidase system. 4. Bors, W ., M. Saran, E. Lengfelder, R. Spottl, and C. MicheL 1974. bMean and SD calculated from four separate samples. The relevance of the superoxide anion radical in biological systems. Curr. Top. Radiat. Res. Q. 9:247-.309. 5. Davis. B. J. 1964. Disc electrophoresis. IL Method and application Milk SOD might be an important naturally-occurring to human serum proteins. Ann. N.Y. Acad. Sci.121:404-427. antioxidant component of milk. In the presence of 6. forman. H. J., and L Fridovich. 1973. On the stability of bovine suitable substrates, the release of superoxide anion in superoxide dismutase. The effects of metals. L Biol. Chern. milk by milk enzymes such as xanthine oxidase or 248:2645-2649. lactoperoxidase, is expected. Other biochemical com 7. Fridovich, l. 1976. Oxygen radicals. hydrogen peroxide and oxygen toxicity. pp. 238-271. In W. A. Pryor (ed.) free radicals in biology, pounds could also generate superoxide anion during VoL I. Academic Press. New York. spontaneous oxidation. If the amount of SOD endogen 8. Goldstein. 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lactoperoxidase. N. Z. J. Dairy Sei. Techno!. 12:37-43. enzymatic function for erythrocuprein (hemocuprein). J. Biol. 12. Holbrook. J., and C. L Hicks. 1978. Variation of superoxide dis Chern. 244:6049-6055. mutase in bovine milk. J. Dairy Sci. 61:1072-1077. 23. McCord. 1. M., and I. Fridovich. 1969. The utility of superoxide 13. Jenness. R., and 1. Koops. 1962. Preparation and properties of a dismutase in studying free radicals reactions. J. Bioi. Chern. 244: salt solution which simulates milk ultrafiltrate. Neth. Milk Dairy J. 6056,6063. 16:153-164. 24. McCord. J. M .. B. B. Keele. Jr .. and I. Fridovich. 1971. An enzyme 14. Johnson. A. H. 1974. The composition of milk. pp. 1-57. In B. H. based theory of obligate anaerobiosis: The physiological function Webb. A. H. Johnson. and J. A. Alford (eds.) Fundamentals of dairy ofsuperoxide dismutase. Proc. Nat). Acad. Sci. 68:1024-1027. chemistry. A vi. Pub!. Co .. Inc .• Westport, Conn. 25. Misra, H. P., and I. Fridovich. 1972. The univalent reduction of 15. Kellogg, Ill, E. W. and I. Fridovich. 1977. Liposome oxidation and oxygen by reduced flavins and quinones. J. Bioi. Chern. 247: erythrocyte lysis by enzymatically generated superoxide and 188-192. hydrogen peroxide. J. Bioi. Chern. 252:6721-6728. 26. Misra. H. P., and L Fridovich. 1972. The generation of superoxide 16. Korycka-Dahl, M., and T. Richardson. 1978. Photogeneration of radical during the autoxidation of hemoglobin. J. Bioi. Chern. superoxide anion in serum of bovine milk and in model systems 24 7:6960-6962. containing riboflavin and amino acids. J. Dairy Sci. 61:400-407. 27. Nishikimi, M. 1975. The generation of superoxide anion in the re 17. Lavelle, F .. A. M. Michelson, and L Dimitrijevic. 1973. Biological action of tetrahydropteridines with molecular oxygen. Arch. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/11/867/1649583/0362-028x-42_11_867.pdf by guest on 02 October 2021 protection by superoxide dismutase. Biochem. Biophys. Res. Biochem. Biophys. 166:273-279. Commun. 55:350-357. 28. Salin, M. L., and J. M. McCord. 1974. Superoxide dismutases in 18. Lowry. 0. H., N.J. Rosebrough, A. L. Farr, and R. J. Randall. polymorphonuclear leukocytes. J. Clin.lnvest. 54:1005-1009. 195 I. Protein measurement with the Polin phenol reagent. 1. Bioi. 29. Schwartz, D. P., and 0. W. Parks. 1974. The lipids of milk: Chern. 193:265-274. Deterioration. pp. 220-274.1n B. H. Webb, A. H. Johnson. and J. A. 19. Marcus, A. J., S. T. Silk, L. B. Safier, and H. L. Gilman. 1977. Alford (eds.) Fundamentals of dairy chemistry. Avi. Pub!. Co., Superoxide production and reducing activity in human platelets. Inc., Westport, Conn. 1. Clin. Invest. 59:149-158. 30. Sutherland, M., and J. M. Gebicki. 1977. The superoxide radical 20. McCay, P. B., and J. L. Poyer. 1976. Enzyme-generated free and singlet oxygen in lipid peroxidation. Austral. Biochem. Soc. radicals as initiators of lipid peroxidation in biological membranes. 10:96. (Abstr.) pp. 238-526. In a Martonosi (ed.) The enzymes of biological mem 31. Weser. G., W. Bohnenkamp, R. Cammack, H.-J. Hartmann, and branes, VoL IV. Plenum Press, New York. G. Voelcker. 1972. An enzymic :-iudy on bovine erythrocuprein. 21. McCord, J. M. 1974. Free radicals and inflammation: Protection of Hoppe-Seyler's Z. Physiol. Chern. 353:1059-1068. synovial fluid by superoxide dismutase. Science 185:529-531. 32. Weser, U., and G. Voelcker. 1972. Evidence for accelerated H 20 2 22. McCord, 1. M., and I. Fridovich. 1969. Superoxide dismutase formation by el)ihrocuprein. FEBS Lett. 22:15-18.

Walker, con'tfromp. 913 institutions. This research also led to Service Corps, he worked as a agencies, continued teaching and the drafting of US legislation to research associate with the Bureau of lecturing at Howard University, protect the right of pre-trial and Environmental Health in Washing George Washington University, and post-trial detainees in jails and ton. Dr. Walker completed his Meharry Medical College of Nash prisons. master's degree in environmental ville. He has contributed numerous Among the developments which health at the University of Michigan articles to professional journals and have taken place in the District of and worked as a public health sani served as a consultant to a number of Columbia food sanitation program tarian, first in Philadelphia, then public and professional organiza while under Dr. Walker's direction Detroit and surrounding areas. Next tions. The recipient of many awards, are amendments of general food he taught at the College of Medicine, Dr. Walker has been honored with regulations, including open dating of Howard University, while beginning the Robert W. Browning prize for products, health examinations of studies in environmental hygiene at disease prevention, outstanding ser employees after illness, weighted Johns Hopkins University. He com vice award of the American Civil inspection forms, four rather than pleted additional studies in water Liberties Union, Special Achieve two inspections per restaurant per supply and pollution control at the ment Award of the American Correc year, display of inspection results in University of Kansas while working tional Food Service Association, sub-standard establishments, and parttime as a research associate in Outstanding Service Commendation identification of fat content of the Environmental Health Research of the U.S. Environmental Protec ground meat. A certificate of merit Laboratory. In 1966 he became a tion Agency, Editor's Award for program was established to motivate research fellow and Director of outstanding contribution to the Jour and reward the food industry and to Environmental Research Laborator nal ofEnvironmental Health, Special inform the public as to which ies for the School of Public Health at Commendation of the U.S. Depart establishments consistently maintain the University of Minnesota where he ment of Justice, Most Distinguished a superior level of cleanliness. completed his Ph.D. Following pub Alumni Award of Kentucky State Dr. Walker's education began in lic health service in Ohio and New University, and Distinguished En Springfield, TN, where he was born Jersey, he assumed his present vironmental Health Scientist Award and grew up. In 1954 he received a position. of the National Environmental B.S. in biology from Kentucky State Throughout other commitments, Health Association. University. Following four years' Dr. Walker has served as a consul service with the Air Force Medical tant to a number of public health