Preparation of Phosphopeptides Derived from αs-Casein and β-Casein Using Immobilized Glutamic Acid-Specific Endopeptidase and Characterization of Their Calcium Binding

Abstract

Phosphopeptides that were derived from αs-CN or β-CN were prepared with immobilized glutamic acid-specific endopeptidase, and their Ca2+ binding was characterized. αs-Casein or β-CN was hydrolyzed in a fluidized bed bioreactor containing 2ml of immobilized glutamic acid-specific endopeptidase by recirculating 20ml of αs-CN or β-CN solution (10 mg/ml in 50mM Tris·HCl and 0.02% NaN3, pH 8.0) for 3h at 20°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 αs-CN hydrolysate and 9.3, 8.2, and 6.2 kDa in the β-CN hydrolysate. The highest concentrations of P were detected in the fractions that eluted with 0.4 and 0.5M KCl for the αs-CN hydrolysate and with 0.4 M KCl for the β-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. αs-Casein phosphopeptides had different patterns for Ca2+ binding than did β-CN phosphopeptides as the total Ca concentration was increased. Calcium binding to these casein phosphopeptides differed from that previously characterized for the tryptic peptides.