Native or denatured protein substrates which are hardly digested by thrombin can become much more efficiently cleaved by the enzyme after chemical modification of their carboxyl groups. Five antibody κ-chains were used to demonstrate this effect. The selective cleavage sites were determined by quantitative N-terminal analysis and N-terminal sequencing. All five κ-chains share the same cleavage sites at Arg-Thr (residues 108–109), Arg-Glu (residues 142–143, Glu side chain modified with glycine amide), Lys-Ser (residues 207–208) and Arg-Gly (residues 211–212). One of the major cleavage sites (Arg-Thr) is located at the joint of the variable/constant region. The amino acids adjacent to these cleavage sites underline the proposed structural requirements for a potential thrombin substrate [(1985) Eur. J. Biochem. 151, 217–224]. This approach can facilitate the application of thrombin in generating large polypeptide fragments of proteins.
