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Journal of Cell and Molecular Biology 9(2): 43-49, 2011 Research Article 43 Haliç University, Printed in Turkey. http://jcmb.halic.edu.tr Cysteine protease from the malaria parasite, Plasmodium berghei- purification and biochemical characterization Emmanuel AMLABU* 1 , Andrew Jonathan NOK 2 , Hajiya Mairo INUWA 2 , Bukola Catherine AKIN-OSANAIYE 3 , Emmanuel HARUNA 4 1 Department of Biochemistry, Kogi State University, Anyigba,Nigeria 2 Department of Biochemistry, Ahmadu Bello University, Zaria,Nigeria 3 Department of Chemistry, University of Abuja, Gwagwalada,Nigeria 4Department of Biochemistry, Kaduna State University, Nigeria (* author for correspondence; ninmac2000@yahoo.com) Rceived: 6 August 2011; Accepted: 22 December 2011 Abstract Plasmodium berghei was isolated from mice red blood cells and phase-separated by Triton X-100 temperature-induced phase separation procedures. The enzyme cysteine protease was purified 5.33 fold with a recovery of 58 %. SDS-PAGE analysis of the enzyme revealed two protein bands with molecular weights corresponding to 18 and 40 kDa, respectively. The enzyme was optimally active at temperature of 40 O C and at a pH of 5.0 (50 mM acetate buffer). Activation energy (6.27 kJ/mole) of the enzyme was determined from Arrhenius plots and initial velocity studies revealed KM and Vmax values of 2.5 mg/ml and 0.2 µmol/min, respectively. The enzyme was inactive on the substrates, albumin and myoglobin. The enzyme was exclusively sensitive to the cysteine protease specific inhibitor iodoacetate (IAA), but was insensitive to Phenylmethylsulphonyl chloride (PMSF), 1, 10 phenanthroline, soybean trypsin inhibitor (SBTI), pepstatin A and EDTA. The synthetic compounds PP-54 and PP-56, currently being evaluated for their anti-malaria potential, competitively inhibited the enzyme activity with corresponding Ki values of 48.88 µg/ml and 0.14 µg/ml, respectively. Keywords: Cysteine protease, Plasmodium berghei, malaria parasite, iodoacetate, enzyme activity Malarya paraziti Plasmodium berghei’den sistein proteaz- saflaştırılması ve karakterizasyonu Özet Fare kırmızı kan hücrelerinden Plasmodium berghei izole edilmiştir ve Triton X-100 uyarılmış sıcaklıkla faz ayırım yöntemleri ile faz ayırımı yapılmıştır. Sistein proteaz enzimi %58 geri kazanımla 5.33 kat saflaştırılmıştır. Enzimin SDS-PAGE analizi sırasıyla 18 ve 40kDa moleküler ağırlıklarına karşılık gelen iki protein bantı ortaya çıkarmıştır. Enzim pH 5.0’te (50 mM asetat tamponu) ve 40ºC sıcaklıkta optimal olarak aktiftir. Enzimin aktivasyon enerjisi (6.27 KJ/mol) Arrhenius grafiğinden belirlenmiştir ve Km ve Vmax değerleri başlangıç hız çalışmaları ile sırasıyla 2.5 mg/ml ve 0.2 µmol/dk olarak belirlemiştir. Enzim albumin ve miyoglobin substratlarında inaktiftir. Enzim özellikle sistein protez spesifik inhibitör iyodoasetata duyarlıdır; fakat PMSF, 1,10 fenantrolin, SBTI, pepstatin A ve EDTA’ya duyarlı değildir. PP-54 ve PP-56 sentetik bileşiklerinin malaryaya karşı potansiyel yarışmalı olarak inhibe edilen enzim aktivitesine karşılık gelen Ki değerleri sırasıyla 48.88 µg/ml ve 0.14 µg/ml olarak ölçülmüştür. Anahtar Sözcükler: Sistein proteaz, Plasmodium berghei, malarya paraziti, iyodoasetat, enzim aktivitesi
44 Emmanuel AMLABU et al. Introduction Malaria remains a tremendous public health burden especially for people living in the tropics, particularly in Africa. About 300-500 million people are infected with the malaria parasite, with up to 1-3 million deaths per year due to the disease (Miller et al., 1994; More, 2002; Martin et al., 2004). The global resistance of malaria parasites to mainstay anti-malarial drugs has intensified the need for the identification of novel chemotherapeutic targets and the development of an effective malaria vaccine. Malaria proteases play distinct roles in the modification of parasite proteins involved in host cell recognition and invasion of red blood cells. Cysteine proteases of parasites have been suggested to have an extracorporeal function in the digestion of host tissues (Rhoads and Fetterer, 1997). Maturing schistosomula and adult schistosomes degrade hemoglobin using cysteine protease for viability maintenance and egg production in the host (McKerrow and Doenhoff, 1988). Plasmodium cysteine protease has been reported to have a critical role in hemoglobin degradation within the food vacuole of Plasmodium falciparum (Rosenthal et al., 1988). Despite the availability of literature on cysteine proteases from malaria parasites, the properties of the enzyme from the aqueous and/or detergent phase(s) of the parasite have not been described. In the present work, we report some properties of the enzyme isolated from the detergent-treated phase of the malaria parasite which can be exploited for precise drug targeting. Materials and methods Materials The compounds (PP-54 and PP-56) were synthesized in India and obtained by Professor A.J Nok and are presently undergoing trials as potential anti-malarials. Other chemicals used in this study were obtained from Sigma, USA. The malaria parasite Plasmodium berghei was obtained from the Kuvin Medical Centre, Hebrew University of Jerusalem Ein keren, Israel. The strain was maintained in our laboratory by serial blood passage from mouse to mouse. Experimental infection A donor mouse with rising parasitemia of 20 % was sacrificed and blood was drawn in heparinized syringe and diluted in phosphate buffered saline. Infection was initiated by needle passage of the parasite preparation from a donor mouse to healthy mice via intraperitoneal route (Peter and Anatoli, 1998; Klemba and Golberg, 2002). Each mouse received 0.2 ml of the diluted infected blood. Course of infection Parasitemia was monitored by microscopic Giemsa-stained thin blood smears. The number of parasitized erythrocytes in about 10-50 fields were counted twice and the average was computed to give the parasitemia of each mouse. Separation of parasite proteins The malaria parasites were phase-separated by Triton X-100 temperature-induced phase separation procedures, using the previously described protocol by Smythe et al. (1990) with slightly modifications. Briefly, 0.5 % Triton X-100 in Tris-buffered saline was added to the parasites and incubated at 4 o C for 90 min. The supernatant was collected after an initial centrifugation at 10,000 x g for 30 min at 4 o C and was layered on 6 % sucrose containing 0.06 % Triton X-100 followed by incubation at 37˚C for 5 min. The aqueous and detergent phases were collected after an initial centrifugation at 900xg for 5 min at 37 o C and were precipitated with cold acetone. The resulting precipitates were referred as the aqueous and detergent phase proteins, respectively. The pellets from each preparation were suspended to 6 ml in 50 mM phosphate buffered saline, pH 7.2 Enzyme activity assays The aqueous and detergent phases of the malaria parasite were used for activity assays by incubating 50 µl of the sample with 500 µl of 100 mM sodium acetate buffer, pH 4.5, and 100 µl of 3 % gelatin. The reaction volume was adjusted to 1 ml with distilled water. Assays were carried out at 37 ºC for an hour and were stopped by the addition of 200 µl of 20 % (v/v) trichloroacetic acid. The precipitated protein was removed by centrifugation (10,000 x g for 5 min at room temperature) and absorbance of the supernatant was read at 366 nm (Dominguez and Cejudo, 1996). One unit of proteolytic activity was defined as 1µmole of tyrosine hydrolyzed per hour under standard assay conditions. Enzyme purification The crude proteins from the detergent-treated phase of the parasite was applied onto a DEAE-cellulose column (1 cm X 12 cm) pre-equilibrated with 50
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