title: Preparation of phosphopeptides derived from alpha s-casein and beta-casein using immobilized glutamic acid-specific endopeptidase and characterization of their calcium binding.
abstract: Phosphopeptides that were derived from alpha s-CN or beta-CN were prepared with immobilized glutamic acid-specific endopeptidase, and their Ca2+ binding was characterized. alpha s-Casein or beta-CN was hydrolyzed in a fluidized bed bioreactor containing 2 ml of immobilized glutamic acid-specific endopeptidase by recirculating 20 ml of alpha s-CN or beta-CN solution (10 mg/ml in 50 mM Tris.HCl and 0.02% NaN3, pH 8.0) for 3 h at 20 degrees C. The molecular masses of casein peptides were monitored by SDS-PAGE. Each hydrolysate was applied to an anion-exchange column using stepwise elution with various concentrations of KCl to separate peptides. The casein phosphopeptide content of the elution profile was monitored by analysis of protein and P concentrations. Calcium binding in phosphopeptide-enriched fractions was determined by CaCl2 titration and measurement of free Ca2+ with a Ca-selective electrode. The electrophoresis patterns showed four major peptides having molecular masses of 10.8, 9.0, 6.6, and 3.6 kDa in the alpha s-CN hydrolysate and 9.3, 8.2, and 6.2 kDa in the beta-CN hydrolysate. The highest concentrations of P were detected in the fractions that eluted with 0.4 and 0.5 M KCl for the alpha s-CN hydrolysate and with 0.4 M KCl for the beta-CN hydrolysate. The calcium-binding ability was found only in the fraction that was eluted with 0.4 M KCl; the maximum Ca2+ binding and the apparent binding constant were 0.24 mmol/mg of protein and 75 M-1, and 0.14 mmol/mg of protein and 148 M-1, respectively. alpha s-Casein phosphopeptides had different patterns for Ca2+ binding than did beta-CN phosphopeptides as the total Ca concentration was increased. Calcium binding to these casein phosphopeptides differed from that previously characterized for the tryptic peptides.
authors.affiliation: Southeast Dairy Foods Research Center, Department of Food Science, North Carolina State University, Raleigh 27695-7624, USA.
authors.forename: O
authors.initials: O
authors.lastname: Park
authors.forename: J C
authors.initials: JC
authors.lastname: Allen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004800:Enzymes, Immobilized; D006868:Hydrolysis; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011189:Potassium Chloride
chemical_list: D002364:Caseins; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D010748:Phosphopeptides; D011189:Potassium Chloride; D010450:Endopeptidases; C075054:glutamic acid-specific endopeptidase, Staphylococcus aureus