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

¹ Southeast Dairy Foods Research Center, Department of Food Science, North Carolina State University, Raleigh 27695-7624

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

Key Words: casein phosphopeptides • immobilized enzyme • glutamic acid-specific endopeptidase • calcium binding

Submitted on August 4, 1997
Accepted on July 13, 1998