Characterization of human kappa-casein purified by FPLC.
Because previous purification procedures for human kappa-casein may have caused the loss of some carbohydrate, relatively gentle methods were used. The protein was isolated by a four-step procedure which included isoelectric precipitation of whole casein, gel chromatography on Sephadex G-200 in the presence of SDS, removal of the SDS with Extracti-Gel D, and FPLC chromatography on Mono Q with buffers containing 6 M urea. The purified protein was nearly identical in amino acid composition to that found earlier by amino acid analysis and peptide sequencing and a molar extinction coefficient of 11.2 +/- 0.1 was determined on the basis of amino acid analysis with a norleucine internal standard. Hydrolysis, acylation, and methylsilylation of the carbohydrate, followed by gas chromatographic analysis on a fused silica column, yielded approximately 5% fucose, 17% galactose, 18% N-acetylglucosamine, 8% N-acetylgalactosamine and 7% sialic acid, totaling almost 55% by weight. The percentages from two different donors were almost the same. About 1 mole phosphorus per mole of kappa-casein was also detected. Using low-speed sedimentation equilibrium methods, a molecular weight of only 33,400 was obtained for human kappa-casein, suggesting carbohydrate lability. Human beta-casein with four phosphoryls was stabilized against precipitation by 10 mM Ca+2 ions at a level greater than 95% when the molar ratio of kappa/beta exceeded 0.15.
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Department of Biochemistry, School of Medicine, Loma Linda University, CA 92350.
Sood
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DeWind
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Slattery
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D000596:Amino Acids / Q000032:analysis; D000606:Amino Sugars / Q000032:analysis; D002118:Calcium / Q000494:pharmacology; D002364:Caseins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D006601:Hexoses / Q000032:analysis; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D010758:Phosphorus / Q000032:analysis; D012794:Sialic Acids / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D000606:Amino Sugars; D002364:Caseins; D006601:Hexoses; D012794:Sialic Acids; D010758:Phosphorus; D019158:N-Acetylneuraminic Acid; D002118:Calcium