Production of anti-peptide antibodies against trypanopain-Tb from Trypanosoma brucei brucei: effects of antibodies on enzyme activity against Z-Phe-Arg-AMC

Anti-peptide Antibodies were produced against the cysteine proteinase trypanopain-Tb from Trypanosoma brucei brucei and the effects of these Antibodies on Enzyme activity against carboxybenzoyl (Z)-Phe-Arg-aminomethylcoumarin (AMC) investigated. A peptide was synthesised corresponding to a region of the trypanopain-Tb active site around the active site histidine so that the resulting anti-peptide Antibodies specifically targeted the active site of the Enzyme. Such Antibodies were considered more likely to modulate Enzyme activity compared with Antibodies directed against other regions of the Enzyme. Trypanopain-Tb activity was modulated by rabbit and chicken Antibodies produced against both the free and conjugated peptide. Rabbit anti-peptide Antibodies enhanced trypanopain-Tb activity by up to 64% at 500 micrograms/ml relative to non-immune Antibodies. Chicken Antibodies on the other hand, both enhanced (by up to 176% at 500 mg/ml) and inhibited (by up to 85% at 250 mg/ml) trypanopain-Tb activity against Z-Phe-Arg-AMC. The nature of the antibody effect depended on the stage during the immunisation protocol at which the Antibodies were produced. Chicken Antibodies also modulated trypanopain-Tb activity in lysates of T.b. brucei, while rabbit Antibodies were only effective against the purified Enzyme. Anti-trypanopain-Tb peptide Antibodies were thus shown to have the potential to affect trypanopain-Tb activity.