Characterization of a hemoglobin-degrading, low molecular weight protease from Plasmodium falciparum.

A protease from Plasmodium falciparum was purified 150 fold by high performance liquid chromatography on a TSK-G-3000 SW exclusion column. The enzyme is not retained during pressure filtration with an Amicon PM10 membrane but is retained by a YM5 membrane. The molecular weight of the protease is less than 10,000, based upon mobility on a calibrated TSK column. The enzyme catalyzes the hydrolysis both of acid denatured hemoglobin and of albumin. The hydrolysis is optimal at pH4.5, but considerable activity is seen at pH 6.0. Pepstatin strongly inhibits the protease (I50 = 70 nM) while bestatin, antipain and phosphoramidon produce moderate inhibition (I50 = 30, 30 and 3 microM, respectively). The protease is inhibited by ferriprotoporphyrin IX (I50 ca. 5 microM). This inhibition is insensitive to pH between pH 4.5 and 6. Although chloroquine does not strongly inhibit the protease, chloroquine-ferriprotoporphyrin IX complex produces inhibition similar to that of ferriprotoporphyrin IX. It is suggested that the antimalarial effect of chloroquine is due to the formation of ferriprotoporphyrin IX-chloroquine complex which prevents the sequestration of ferriprotoporphyrin IX into malarial pigment, thereby providing both ferriprotoporphyrin IX and its chloroquine complex as inhibitors of one of the proteases required for the degradation of hemoglobin.