Proteolytic specificity of chymosin on bovine α s1 -casein

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.