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Isolation and characterization of a cysteine proteinase from Fasciola hepatica adult worms

Isolation and characterization of a cysteine proteinase from Fasciola hepatica adult worms

Molecular and Biochemical Parasitology, 351: 89-95

Adult Fasciola hepatica worms contain multiple proteinases capable of degrading hemoglobin, immunoglobulins and collagen. Here we report the isolation and biochemical characterization of a cysteine proteinase from acidic extracts of these worms. The enzyme was purified to homogeneity by cation exchange and molecular sieve high-performance liquid chromatography. It eluted at a native molecular weight of approximately 14,500 and migrated as a single band at approximately 14,500 Da upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity was assessed by employing synthetic peptide substrates, such as carbobenzoxy-phenylalanyl-arginyl-7-amino-4-trifluoro-methylcoumarin, commonly used to assay other cysteine proteinases. The proteinase was maximally active at pH 6.0, with 50% or more of the activity detected between pH 4.5 and 7.5. Inhibition of activity at pH 5.5 was seen only with compounds known to inhibit cysteine proteinases. No effect was seen with inhibitors of aspartic, serine, or metalloproteinases. The purified enzyme was stable at acidic pH at 4 degrees C, 25 degrees C, -20 degrees C, and in 1 M urea.

Accession: 001871368

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PMID: 2761575

DOI: 10.1016/0166-6851(89)90146-1

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