Monoclonal antibody studies of creatine kinase. Antibody-binding sites in the N-terminal region of creatine kinase and effects of antibody on enzyme refolding

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Abstract
(1) The binding sites of two monoclonal antibodies, CK-2A7 and CK-5H5, have been located to a 60-amino-acid sequence in the N-terminal region of creatine kinase (CK) by the use of chemical cleavage with formic acid (which cleaves proteins at Asp-Pro bonds) and cyanogen bromide (which cleaves at Met residues). (2) A simple method for preparing chemically-cleaved fragments of proteins for electrophoresis and Western blotting is described. (3) Binding studies with CK preparations from different animal species show that single amino acid changes at residues 39 or 82 prevent binding of CK-2A7 and CK-5H5 respectively. We suggest that Lys-39 and Glu-82 form parts of the binding sites on CK for the two monoclonal antibodies. The two sites lie in variable regions at each end of a highly-conserved sequence (residues 46 to 79) and are inaccessible to antibody in the native enzyme. (4) One of the antibodies, CK-2A7, inhibits the refolding of CK to native enzyme after denaturation by urea.