Antibodies to rat procathepsin B recognize the active mature enzyme

Use of mature cathepsin B for immunization invariably yields antisera that react with the denatured protein but not with the native enzyme. This is thought to be due to spontaneous denaturation of the immunizing antigen on introduction into the animal. Recombinant rat procathepsin B has been expressed in yeast as a secreted product. A procathepsin B mutant (Cys29Ser), where autoprocessing is prevented, has been purified and used to raise a rabbit polyclonal antiserum. Both immunodiffusion analysis and an activity depletion assay demonstrated that this antibody recognized native mature cathepsin B. It appears that conformational epitopes existing on the active enzyme are lost on denaturation. The stability of the proenzyme however permits their presentation for antibody generation.