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Hekken & Thompson, 1992. Application of PhastSystemB to the Resolution of Bovine Milk Proteins

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Electrophoresys aplied to bovine milk proteins determination
  Application of PhastSystemB to the Resolution of Bovine Milk Proteins on Urea-Polyacrylamide Gel Electrophoresis D. L. VAN HEKKEN and M. P. THOMPSON Agricultural Research Service, USDA Eastern Regional Research Center Philadelphia, PA 19118 ABSTRACT Optimal conditions were established for alkaline urea-PAGE using modified precast, ultrathin gradient gels on the automated PhastSystem@. Profiles of milk proteins showed that the caseins and whey proteins resolved extremely well. Major bands were observed for %,-casein and &casein, and %-casein appeared as a well-resolved doublet. In contrast, K-casein separated from other caseins as a faint doublet, and purified K- casein appeared as one major and one minor band. Whey proteins (serum albu- min, a-lactalbumin, ~lactoglobuh) ep- arated into broad bands resolved from each other and from the caseins. Partially (40%) dephosphorylated whole casein showed multiple bands for %I-casein and pcasein at different levels of phos- phorylation. Separation of genetic pheno- types was observed for lactoglobulin A and B; &lcasein A, B, and C; and P- casein A, B, nd C. Electrophoretic pat- terns of milk proteins extracted from cheese samples varied among the differ- ent types of cheeses. Our modified pro- cedure provides researchers with a rapid technique to separate both caseins and whey proteins on the same urea gel ac- cording to their charge to mass ratios. (Key words: casein, PhastSystem@, urea-electrophoresis, whey proteins) Abbreviation key: BSA = bovine serum albu- min; CN = casein, used with &I-, z-, p- and K-; LA = lactalbumin, LG = lactoglobulin; and Rm = relative migration. Received September 18. 1991. Accepted January 6, 1992. 'Mention of brand or fm ames does not constitute an endorsement by the US Department of Agriculture over others of a similar ~hlre ot mentioned. INTRODUCTION Polyacrylamide gel electrophoresis is an ex- cellent research tool for the identification of milk proteins because proteins can be sepa- rated according to size (1) or charge to mass ratio (8, 9). The four caseins that make up whole casein (CN) ave similar molecular weights (19,OOO to 25,230) and distinct differ- ences in their amino acid sequences and their degree of phosphorylation (1,5, 8 to 9, and 10 to 13 phosphate groups per molecule for K-CN, P-CN %l-CN, and a&ZN, respectively) (3). Alkaline PAGE uses this difference in charge to mass ratio to resolve the individual caseins. Incorporation of urea into the samples and the use of 7 acrylamide slab gels permit the separation of most casein phenotypes (7, 9, lo), detect the removal of phosphate groups from individual caseins (2). and monitor the proteolysis of casein in aging cheeses (4, 5). Electrophoretic separation of whey proteins us- ually requires native (without urea alkaline PAGE (7, 9, 10) because the use of urea results in smeared, unresolved whey proteins (7) and poor resolution of a-lactalbumin (LA) from q-CN and P-lactoglobulin (LG) from Slab gels am large (12 x 15 x .5 cm) and usually require many hours to separate the proteins and to stain and destain protein bands. Recent developments in automated electropho- retic equipment and the commercial availabil- ity of precast, ultrathin, miniature gels allow for the resolving, staining, and destaining of proteins within a few hours. The Phast- System@ (Pharmacia, Uppsala, Sweden) has been used for SDS-PAGE 6), isoelectric focusing (13) of caseins, and identification of dephosphorylated caseins (12). The goal of this study was to select urea- PAGE conditions for the PhastSystem@ that would resolve caseins according to charge to mass ratio and permit the simultaneous resolu- tion of casein and whey proteins. p-CN (10). 1992 J Dairy Sci 75:1204-1210 1204  PTSYSlEM@ ON MILK F ROTBINS 1205 MATERIALS AND METHODS Materlals The materials and their sources used in this study were as follows: a-LA and bovine serum albumin BSA) from Sigma Chemical Com- pany (St. Louis, MO); b-LG rom Pentex Bio- chemical (Kankakee, E ; -mercaptoethano1, Phast@ buffer strips, and PhastGels@ from Pharmacia LKB Biotechnol. Inc. (Piscataway, NJ); and potato acid phosphatase (59 units/ml at 25 C, pH 4.8) from Calbiochem Corp. (La Jolla, CA). Samples obtained from within the Eastern Regional Research Center included Mozzarella cheese from Michael Tunick; ren- net K-CN from Myth Malin; and PLG A and B and caseins containing q1-C A, B, and C and b-CN A, B, and C from Harold Farrell, Jr. processed American, Danish Blue, Cheddar, creamed cottage, and Ricotta cheeses were purchased locally. Sample Sources Skim milk, prepared from pooled raw milk from a commercial herd, was dialyzed exten- sively against deionized, distilled water at 4'C to remove lactose and salts and then lyophi- lized. Sodium caseinate was prepared as fol- lows: skim milk was acid-precipitated at pH 4.6 and 20'C using W HCl and centrifuged at 6000 x g for 15 min at 20°C. The precipitate was dissolved in water adjusted to pH 7.0 with 1 N NaOH. The acid-precipitating, centrifug- ing, and dissolving steps were repeated. The casein solution was dialyzed ovemight and Partially dephosphorylated whole casein was obtained according to Van Hekken and Strange (11). An aqueous solution containing 2.5 mg of whole casedml and .065 units of potato acid phosphatase/ml at pH 6.5 was in- cubated in a shaking water bath (37'C, 150 rpm) for 60 min. The solution was heated at 80°C for 5 min to inactivate the enzyme; dia- lyzed for 36 h in 4'C distilled, deionized wa- ter; and lyophilized, The procedure of Zittle and Custer (14). involving sulfuric acid and ammonium sulfate precipitation and ethanol purification, was used to extract K-CN from wet acid casein. The protein was dialyzed for 5 d and lyophilized. lyophilized. Cheese samples (5 g) were chopped finely with a knife except for cottage and Ricotta, the soft curds of which did not require mechanical disruption. All cheeses were then extracted three times with acetone (100 ml) to remove fat and water. The wet cheese SNF were Msed once in diethyl ether and air dried. Urea-PAGE Urea-PAGE with the PhastSystem@ was based on Pharmacia's native PAGE separation technique (file number 120) as modified for caseins by Van Hekken et al. (12) and described in det il herein. All of the gels avail- able through Pharmacia were tested, and the best resolution of milk proteins was obtained on the 8 to 25 gradient gels. Prior to use, native miniature (50 x 43 x .45 mm) gels were modified by soaking for 15 min in 6.6 M urea, .112 M Tris, .112 acetate buffer at pH 6.4 and air dried for 3 to 5 min. Native buffer strips containing .88 M Galanine and .25 M Tris at pH 8.8 were used. Samples (8 to 100 pgIp1) were dissolved in the urea-Tris-acetate buffer with 10 2-mercaptoethanol and .025 bromophenol blue. Approximately 1 pl of sample was deposited in each lane. Electropho- retic conditions programmed into the Phast- System@ were 300 V, 7.5 mA, 2.5 W, t 15°C for 10 Vh (volthour). Samples were applied at 5 mA until 2 Vh were attained, and then the setting was returned to initial values. Gels were run for 110 accumulated Vh (approxi- mately 75 min). Running at V and mA settings lower than suggested by Pharmacia was re- quired to prevent a-LA and l-CN from migrating too close to the leading edge. Pro- tein bands were stained for 8 to 10 min at 50 C in .25% Coomassie brilliant blue R dissolved in 301060 methano1:acetic acid water (Phast- System@ development technique file number 200). The gels were destained in 30:10:60 methano1:acetic acidwater until the back- ground was clear. Relative migration (Rm) rates were calculated as described by Thomp- son (1 0) and were based on a minimum of five runs. The distance from the srcin to the slower band of the cx N doublet was given a value of 1.00. The migration distances of all other protein bands were measured relative to this standard, Journal of Dairy Science Vol. 75, No. 5, 1992  1206 VAN HElUCEN AND THOMPSON Origin BSA K-CN j3-CN aS2-CN P-LG a,,-CN a-LA Lane 12345678 Figure 1. Urea-PAGE pmfde of bovine caseins and whey pmteius using 8 to 25 gradient gel nd the PhastSystem@. Samples in lanes are 1) whole casein (CN), ) K-CN, 3) para-~-CN, ) 40 dephosphorylated whole casein 5) skim milk 6) ~lactalbumh a-LA), ) J ctoglobulin @LG), and 8) bovine serum albumin (BSA). RESULTS AND DISCUSSION Electrophoresis Urea-PAGE using large slab gels has disad- vantages in the lengthy running time required for the resolution of caseins and in the wide variety in procedures used among different laboratories to obtain profiles (8,9). Use of the PhastSystem* significantly decreased the time and quantity of materials required to obtain quality profiles and allows for the elecmphe retic conditions to be programmed in advance Id f Dairy Science Vol. 75, No. 5, 1992 and automatically carried out. The availability of standardized and prepared gels and buffer strips reduced variation because of methodol- ogy This should encourage direct comparisons of gel profiles among laboratories. Caseln and Whey Profiles Caseins were the best resolved of all the milk proteins when examined on urea-PAGE (Figure 1). The profiles for whole casein and skim milk (lanes 1 and 5, respectively) had - CN and p-CN bands typical of urea-PAGE (9,  PHASTSYSTEh@ ON MILK PROTEENS 1207 IO, 12). In whole casein and skim milk pro- files, K-CN appeared as a very faint doublet that was not as sharply resolved as the other caseins. purified K-CN (lane 2) had one major and one minor band. The faster moving minor The only band for the rennet K-CN sample (lane 3) appeared above the srcin and faded rapidly during destaining. Rennet treatment converts K-CN to puru-~- SN, which, under alkaline conditions, migrates to the cathode (4). Various other minor caseins were also noted in the whole casein and skim milk sam- ples. purified whey proteins, a-LA, &LG, and BSA (lanes 6,7, nd 8, respectively), migrated in broad bands that, although not as sharp as the casein bands, were easily identified. The whey proteins were purchased as purified pro- band was thought to be glyCOSylated K CN. teins, and each contained minor contaminants. As seen in skim milk (lane 5), the whey pro- teins and caseins resolved well from each 0th- er. Past attempts to observe both groups of milk proteins on the urea-PAGE slab gels typi- cally resulted in streaking of whey proteins 0) or the inability to separate a-LA from h1 m and P-LG from P-CN IO). Our success in separating both groups of proteins may have been due to 1) the use of urea, which disas- sociated the proteins and separated proteins according to net charge, and the presence of a gradient within the gel, which resolved a-LA and %l-CN, and &LG and p-CN by molecular sieving; and due to 2) rapid separation times that minimized diffusion of the sample in the gel. Resolution according to charge to mass ra- tio has been the major reason for using urea- rgin ] P-CN A,B and C ] a,,-CN A,B, and C p-LG A and B [ Pip . Urea-PAGE rofile of bovine whey proteins and caseins using 8 to 25 went gel and the PhastSystem@. Samples in anes contain 1) iMactoglobulin PLG) A, 2) BLG B, ) i-casein (%l-CN) A, 4) l-CN B ) l-CN C, ) &casein @CN) A, 7) whole uein containing (3CN A/B, and 8) fL04 C. Variant mi proteins, in order of migration rates (highest fmt), resulted in the following order: A, B, and C. Journal of Dairy Science Vol. 75. No. 5. 1992
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