Biotechnol. Appl. Biochem. (2009) 52, 149–157 (Printed in Great Britain) doi:10.1042/BA20080015 149 Generation of an affinity matrix useful in the purification of natural inhibitors of plasmepsin II, an antimalarial-drug tar Angel R. Ram ´ ırez* 1,2 , Yasel Guerra*, Anabel Otero*, Beatriz Garc ´ ıa* 3 , Colin Berry†, Judith Mendiola‡, Aida Hern ´ andez-Zanui§ and Mar ´ ıa de los A. Ch ´ avez* 1 *Centro de Estudio de Prote ´ ınas, Facultad de Biolog ´ ıa,Universidad de la Habana, Calle 25 #455 entre J e I, Vedado, CP 10 400, La Habana, Cuba, †Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, Wales, U.K., ‡Instituto de Medicina Tropical ‘Pedro Kour ´ ı’,Autopista Novia del Mediod ´ ıa,km 6 1/ 2 entre Carretera Central y Autopista Nacional, La Lisa, La Habana, Cuba, and §Instituto de Oceanolog ´ ıa,CITMA, Ave 1ra y 196, Playa, La Habana, Cuba An affinity matrix containing the antimalarial drug target Plm II (plasmepsin II) as ligand was generated. This enzyme belongs to the family of Plasmodium (malarialparasite)asparticproteinases, known as Plms (plasmepsins). The procedure established to obtain the support has two steps: the immobilization of the recombinant proenzyme ofPlm II to NHS (N-hydroxysuccinimide)-activated Sepharose and the activation of the immobilized enzyme by incubation at pH 4.4 and 37 ◦ C. The coupling reaction resulted in a high percentage immobilization (95.5 %), and the matrices obtained had an average of 4.3 mg of protein/ml of gel. The activated matrices, but not the inactive ones, were able to hydrolyse two different chromogenic peptide substrates and haemoglobin. This ability was completely blocked by the addition of the general aspartic-proteinase inhibitor, pepstatin A, to the reaction mixture. The matrices were useful in the affinity purification of the Plm II inhibitory activity detected in marine invertebrates, such as Xestospongia muta (giant barrelsponge)and the gorgonian (sea- fan coral) Plexaura homomalla (black sea rod), with increases of 10.2-and 5.9-fold in the specific inhib- itory activity respectively. The preliminary K i values obtained, 46.4 nM (X. muta) and 1.9 nM (P. homomalla), and the concave shapes of the inhibition curves reveal that molecules are reversible tight-binding inhibitors of Plm II. These results validated the use of the affinity matrix for the purification of Plm II inhibitors from complex mixtures and established the presence of Plm II inhibitors in some marine invertebrates. Introduction Malaria is still the most serious parasitic disease affecting humankind. The WHO (World Health Organization) estim- ates that between 350 and 500 million infections occur every year. The disease is most prevalent in tropical and subtropical areas ofthe planet, where up to 2.7 million people die annually, mostly children less than five years old [1–3]. Four species of parasites of the genus Plasmodium cause malaria in humans, and Plasmodium falciparum is responsibl for the most malignant form of the disease. This malarial parasite species is highly adaptable by way of mutation, whic has led to the appearance of resistance to antimalarial drugs The re-emergence of the disease as a major public-health problem has produced the urgent need for new antimalarial compounds active against previously identified drug targets [1–3].Potentially interesting are the aspartic proteinases, known as Plms (plasmepsins), some of which are involved in haemoglobin degradation. The inhibition of these enzymes leads to parasite starvation in vitro [4–6]. Plm II is the best characterized Plm, and several inhibitors of this enzyme have already been developed using both rational design and chemical library screening, although none are yet suitable for drug use [7,8]. Interestingly, there are only a few reports of Plm II inhibitors in natural sources [9–11].The isolation ofPlm IIinhibitorsfrom complex mixtures, such as crude extracts of living organisms, require the establishment of reliable, selective and fast purification techniques. For this purpose,we developed an affinity chromatography matrix, through the immobilization of the P.falciparum proPlm II recombinant zymogen. The matrix was validated by the partial purification of Plm II inhibitors Key words: antimalarial-drug target, Plasmodium (malarial parasite), plasmepsin II inhibitor, Plexaura homomalla (black sea rod), Xestospongia muta (giant barrel sponge). Abbreviations used: ACN,acetonitrile; NHS,N-hydroxysuccinimide; Plm,plasmepsin; RP-HPLC,reverse-phase HPLC; TFA,trifluoroacetic acid. 1 Correspondence may be addressed to either of these authors (email aramirez@fam.cug.co.cu or mchavez@infomed.sld.cu). 2 Present address: Facultad Agroforestal de Montana, Centro Universitario de Guant ´ anamo, Guant ´ anamo, Cuba. 3 Present address: Department of Biological Sciences, 2500 University Drive N.W.,University of Calgary, Calgary, AB,Canada T2N 1N4. C 2009 Portland Press Ltd