Thrombin specificity

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Abstract
Selective cleavage of polypeptides by α-thrombin can be reasonably predicted [Chang, J.-Y. (1985) Eur. J. Biochem. 151, 217–224]. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse k light chains 10K26 and 10K44 from anti-(azobenzene arsonate) antibodies contain 20 Arg/Lys-Xaa bonds. Only two of them, one ProArg-Thr bond located at the joint of the variable region with the joining peptide and one ValLys-Ser bond located near the carboxyl-terminal end of the constant region, were selectively cleaved by α-thrombin. The ProArg-Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys-Ser has a 50% cleavage time approx. 9–10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short-time thrombin digestion. Fragments containing intact variable region (1–96) and intact joining peptide–constant region (97–214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) λ light chains from both human and mouse all begin with the GlnProLys-(Ala/Ser) structure (positions 108–111) at their constant regions. This ProLys-Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human λ chain (KERN, NEI, NEW, VOR) and one mouse λ chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys-Ala/Ser bonds. For human λ chains, the 50% cleavage time at this ProLys-Ala bond was approx. 3–4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC-20. (c) Heparin inhibits thrombin cleavage of Arg/Lys-Xaa bonds located near the center of the antibody light chain, but slightly activates thrombin cleavage of those located near the amino or carboxyl-terminal ends of the protein. The significance of these findings is threefold. (a) It demonstrates that selective cleavage of large polypeptides by α-thrombin can also be reasonably predicted. (b) It provides a useful method for light chain fragmentation which can greatly facilitate amino acid sequencing of antibodies. (c) It serves to generate fragments containing intact variable regions and constant regions from antibody light chains of human and mouse. Such fragments may be useful for chemical semisynthesis of a human-mouse light chain chimeras.

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