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Superoxide Dismutase Activity in Bovine Milk Serum

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Superoxide Dismutase Activity in Bovine Milk Serum 867 Journal ofFood Protection Vol. 42, No. 11, Pages 867-871 (November, 1979) Copyright© 1979, International Association of Milk, Food, and Environmental Sanitarians Superoxide Dismutase Activity in Bovine Milk Serum 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, ;<X; xg for 5 min i I I ~ I I ~ L~-~'- ... L .... ~"-c--__j__-~=--__;.-~L. ---"---="-' 200 400 600 800 1000 1ONE UNIT OF ENZYME ACTIVITY SUPEROXIDE DISMUTASE (ng/mD Figure 2. Inhibition of cytochrome c reduction by purified bovine Crude J:.superoxide diamutase erythrocyte superoxide dismutase (SOD). Superoxide anion was preparation generated by the xanthine-xanthine oxidase reaction. Each point on the Figure 1. isolation of'superoxide dismutase <SOD )from bovine milk. curve represents an average of two measurements, each in duplicate. Simulated milk ultrafiltrate was prepared as described by Jenness and Bars represent upper and lower limits ofvalues. Koops (13). Only that part ofthe curve where the percent inhibition of cytochrome c A1ilk serum preparation reduction was directly proportional to SOD concentration was used to calculate the amount of SOD present in milk. Samples of raw bovine milk were obtained from the University of Wisconsin dairy herd and contained milk pooled from several breeds of Assay of'SOD activity on polyacrylamide gels cows. The milk was cooled to 4 C and used the same day for Samples of purified bovine erythrocyte SOD or the crude preparation preparation of milk serum as follows. The skim milk fraction obtained of bovine milk SOD were dialyzed against Tris-glycine buffer. pH 8.3 by centrifugation at 1000 x g (10 min, 25 C} was further centrifuged at for 24 h at 4 C. The retentate obtained after dialysis was diluted with 100,000 x g (60 min, 4 C). The 100,000 x g supernatant fluid containing the same buffer to the desired enzyme concentration, dissolved in a the milk serum fraction was then dialyzed for 6 h against 25 volumes of sucrose solution (40% w/v) and applied onto the polyacrylamide gels. 0.05 M potassium phosphate buffer, pH 7.8 at 4 C. Retentate from The gels were prepared and the samples subjected to disc dialyzed milk serum wa> used for assay of SOD activity. electrophoresis according to the procedure of Davis (5). Gels were stained for SOD activity with N BT in the presence of photogenerated Assay o/SOD activity. superoxide anion (2). The photogenerated superoxide anion reduced The assay was based on the ability of SOD to dismutate superoxide the dye to insoluble blue diformazan except in those areas where SOD anion and thereby to specitlcally and efficiently compete with electron destroyed the radical, resulting in formation of achromatic bands. acceptors for superoxide radicaL SOD activity in milk serum was measured spectrophotometrically from the amount of inhibition of the Protein assay reduction of cytochrome c by the superoxide anion produced Protein concentrations were assayed in milk serum or partially­ enzymatically in the xanthine-xanthine oxidase reaction: purified SOD preparations by the method of Lowry et al. (18) using bovine serum albumin or pure SOD protein as standards. respectively. 3 2 2 Cyt e (Fe + l + 20T ------;;,. 2 Cyt c (Fe + ) + 201 RESULTS AND DISCUSSION The assay for enzyme activity was based on the 2~J~~D l capacity of SOD to inhibit reduction offerricytochrome c Absorbance at 550 nm by superoxide anion. Since ascorbic acid present in milk serum might interfere with the assay, it was removed by The conditions for the assay were similar to those described by Weser and Voelker \32). The reaction was carried out at 25 C in a cuvette dialysis (MW cutoff ca. 14,000) against 0.05 M potassium \l·cm light path) in a tina] volume of l ml (air-equilibrated) containing phosphate buffer (pH 7.8 at 4 C) before the analyses were SO mM potassium phosphate buller. pH 7.8. 0.33 mM xanthine, started. 0.027 mM ferricytochrome c. 0.1 mM EDTA. 16 nM catalase and As indicated in Fig. 3, retentate from dialyzed milk 210 nM xanthine oxidase. A similar solution without xanthine oxidase serum possessed the same qualitative inhibitory activity served as the blank. Cytochrome c reduction was measured at 550 nm in a Beckman Acta Ill double-beam spectrophotometer as a as purified bovine erythrocyte SOD in the assay of function of time. The qtochrome c reduction reached a plateau after cytochrome c reduction by superoxide anion generated in 20 min.
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