Effect of calcium on thermolysin hydrolysis of β-casein tryptic peptides

Bovine beta-casein was hydrolyzed with trypsin in the presence of 2 mm NaCl, the hydrolysate was centrifuged and the supernatant was ultrafiltered on a 10-kDa membrane. Under these conditions, mainly the peptides beta-CN 1-25 and beta-CN 33-97 A(1) and A(2) were produced as determined by RP-HPLC and IES-MS analyses. This hydrolysate was further submitted to thermolysin hydrolysis in the presence of NaCl (2 mm) or CaCl2 (0.2 and 2 mm). The results revealed accelerated hydrolysis by thermolysin of the peptide beta-CN 1-25 in the presence of calcium ions (2mm), while the degradation of the peptide beta-CN 33-97 A, and A2 was affected by the presence of neither NaCl nor CaCl2. The effect of calcium on the rate of proteolysis of the peptide beta-CN 1-25 might be related to the binding of calcium to the phosphoseryl cluster, inducing structural changes in the peptide, and the affinity of thermolysin for specific peptidic bonds, in particular, for the peptidic bonds Ser(22)-Ile(23) and Glu(11)-Ile(12). Binding of calcium ions to the phosphoseryl residues of proteins could provide a means of controlling the release of specific peptide sequences during proteolysis. (C) 2003 Elsevier Ltd. All rights reserved.