Cystatin‐like cysteine proteinase inhibitors of parasitic protozoa

A new method has been developed for detecting cystatins and other cysteine proteinase inhibitors. The method, which involves protein separation by SDS‐PAGE followed by a cysteine proteinase overlay step, is more sensitive than previously reported techniques: as little as 1 ng of recombinant human cystatin C can be detected and cysteine proteinase inhibitors could also be detected in complex protein mixtures such as bovine foetal serum. The method has been used to show, for the first time, cysteine proteinase inhibitors in lysates of a range of parasitic protozoa (Trypanosoma brucei, Leishmania mexicana mexicana, Toxoplasma gondii and Tritrichomonas foetus) and to confirm that one occurs in the free‐living ciliate Tetrahymena pyriformis. Cystatin‐like inhibitory activity was also demonstrated in boiled lysates of L. mexicana mexicana using conventional assays methods.