Proteolytic specificity of chymosin on bovine αs1-casein

Abstract

Summary: In dilute buffers ⋟ p^{H 5·8}, chymosin hydrolysed bovine α_s1-casein to α_s1-I, α_s1-II and α_s1-III/α_s1-IV in a sequential manner while at p^{H 4·6} α_s1-casein was hydrolysed to α_s1-I which was then hydrolysed to α_s1-V. In the presence of 5 % (w/v) NaCl at p^{H 5·2}, α_s1-casein was hydrolysed to α_s1-I which was then hydrolysed to α_s1-VII and α_s1-VIII. α_s1-I, α_s1-II and α_s1-III/α_s1-IV were isolated by chromatography on cellulose phosphate followed by preparative slab-gel electrophoresis; α_s1-V was isolated by repeated preparative slab-gel electrophoresis and α_s1-VII by gel filtration on Sephadex G-150 followed by preparative slab-gel electrophoresis. The mol. wts of the peptides, estimated by gel filtration on Sephadex G-100, were 21000, 17600, 15600, 19900 and 14600 for α_s1-I, α_s1-II, α_s1-III/α_s1-IV and α_s1-V and α_s1-VII respectively. Characterization of the peptides by amino acid, phosphorus and terminal residue analysis showed that they probably consisted of segments of the α_s1-casein chain as follows: α_s1-I: residues 24/25–199; α_s1-II: residues 24/25–169; α_s1-III/α_s1-IV: residues 24/25–149–150; α_s1-V: residues 29/33–199; α_s1-VII: residues 56–179. Peptide bonds close to phosphate residues on the α_s1-casein chain were not hydrolysed by chymosin at high p^H values (⋟ 5·8) when the phosphate groups were charged, but became available for hydrolysis when the reaction pH was reduced. Proteolytic specificity was also modified by NaCl.