Purification and partial characterization of an acid proteinase from Dirofilaria immitis

An acid proteinase of Dirofilaria immitis worms was purified 437-fold by gel filtration on Sephadex G-75 followed by pepstatin-agarose gel affinity chromatography. The enzyme with a molecular weight of 42,000 and specific activity of 384 units (mg protein)-1 was homogeneous as judged by both affinity chromatography and SDS-polyacrylamide gel electrophoresis. However, polyacrylamide disc electrophoresis at pH 8.9 revealed that the enzyme is composed of 5 multiforms, all carrying proteinase activity. Optimum pH of the enzyme was in the range of pH 2.8-3.4, and its isoelectric point ranged between 5.8 and 6.4. The purified proteinase showed a potent activity against hemoglobin and myoglobin releasing acid soluble peptides, but not free amino acids. Complete inhibition of the proteolytic activity by pepstatin in the order of 10(-7) M strongly suggests that the enzyme belongs to the carboxyl-proteinase family.