Partial Purification and Evaluation of Substrates-preference of Cysteine Protease from Plasmodium berghei
Abstract
Malaria, a disease caused by a parasitic protozoan of the phylum apicomplexan, remains an important disease of the
tropics particularly Africa, where pregnant women and children constitute most affected population. Plasmodial cysteine
proteases play important roles in the progression of malaria and have constituted key chemotherapeutic targets for screening
potential antimalarial compounds. This work was designed to evaluate the success of two strategies for possible release of
cysteine protease from Plasmodium berghei isolates and to partially purify the enzyme molecule. Parasite isolates were prepared
from the blood of 25 infected mice at high parasitaemia (~ 24-29%) using saponin treatment. Release of the crude soluble
enzyme from the parasite isolates using freezing and thawing and Triton-X100 treatments were compared. The enzyme assay
was carried out by incubating the enzyme with 3% gelatin (as substrate), at pH 5.5 and 37oC for 60 minutes terminated using
40% trichloroacetic acid. The enzyme activity was taken as the amount of tyrosine (measured at 260 nm) released in 60 minutes.
Enzyme assay revealed significantly higher enzyme activity in Triton-X100 fractions (TX1) than in frozen and thawed fractions
(p < 0.05). Dithiothreitol (DTT) and cysteine displayed varying stimulatory actions on the soluble enzyme. Variation in the
preference of TX1 for various peptide substrates was observed, with maximum activities recorded with Gelatin after incubation at
37oC for 60 minutes. The pooled TX1 fractions (~16ml) were partially purified by ammonium sulfate precipitation (80%
saturation), dialysis and gel filtration (Sephadex-G200) resulting in purification of approximately four fold and 82.0% yield.
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