In hemozoin

Another possible participant in hemozoin nucleation is the histidine-rich protein (HRP). Using monoclonal antibodies to probe the proteins of the digestive food vacuole of P. falciparum, Sullivan et al. identified two histidine-rich proteins, HRP II and HRP III, and demonstrated that these proteins could mediate the formation of hemozoin [37]. HRP II (Mr 35 kD) contains 51 repeats of the sequence Ala-His-His (76% of the mature protein is histidine and alanine) [38], while HRP III (Mr 27 kD) contains 28 Ala-His-His sequences (56% of the mature protein is histidine and alanine) [39]. While a clone lacking both HRP II and III has been shown to produce hemozoin, a third protein which cross reacted with anti-HRP monoclonal antibodies has also been found in the digestive vacuole of this strain. Although this protein has not been isolated or shown to mediate hemozoin formation, a candidate is HRP IV (Mr 10 kD) which is 31% histidine [40]. 

Digestion of Hb and heme detoxication in hemozoin appears to be very critical steps in the intraerythrocytic cycle. By providing necessary amino acids to the parasite [16], the digestion of Hb is crucial for equilibrium of osmotic pressure in the parasitized RBC in order to prevent a premature lysis before the completion of the cell cycle [17]. Such processes also provide space for the parasite to increase in size. If about 70-80% of the host cell Hb is digested by the parasite [18], only about 16% of produced aminoacids are used for protein synthesis [16, 19]. 

Figure 11.7 Heme groups in different binding situations (a) free heme which is cell toxic (b) In hemoglobin, the heme is bound to a protein backbone (R). Hemoglobin is ordered in a quaternary structure, meaning that four globular a-helical subunits arrange in a tetrahedral shape, (c) Heme is detoxified in hemozoin crystals, which are insoluble.

Hemozoin, also known as malaria pigment, is, in teims of its chemical composition, identical to (3-hematin. Hemozoin is formed as a crystallization product of heme under the acidic conditions present in the food vacuole of malarial parasites. In the crystal, the heme molecules are linked into dimers through reciprocal iron-carboxylate bonds to one of the propionate side chains of each porphyrin. The dimers form chains linked by hydrogen bonds. 

LDMS is particularly well suited for the analysis of porphyrins.35-39 The heme molecule—a 22 rc-electron conjugated protoporphyrin system (Figure 8.1)—is an efficient photo-absorber in the visible and near UV (with an absorption maximum—the Soret band—near 400nm). This feature, concurrently with its low ionization potential, warrants that direct LDMS will possess extremely low limits for heme detection. The uses of IR or UV LDMS for structural characterization of natural porphyrins and their metabolites, synthetic monomeric porphyrins (e.g., used in photodynamic therapy), porphyrin polymers, and multimeric arrays, have been well documented.41148 In addition fast atom bombardment MS has been used to characterize purified hemozoin, isolated from the spleens and livers of Plasmodium yoelii infected mice.49... 

Loup C, Lelievre J, Benoit-Vical F, Meunier B. (2007) Trioxaquines and heme-artemisinin adducts inhibit the in vitro formation of hemozoin better than chloroquine. Antimicrob Agents Chemother 51 3768-3770. 

Chloroquine probably acts by concentrating in parasite food vacuoles, preventing the biocrystallization of the hemoglobin breakdown product, heme, into hemozoin, and thus eliciting parasite toxicity due to the buildup of free heme. 

While there is no doubt that free lipids can facilitate the formation of hemozoin in model systems, their potential biological role must be placed in the appropriate context. The vast majority of these lipids are involved in cellular structures (organism membrane, organelles, etc.), not freely soluble in the cytoplasm. The methods of extraction modified from Bligh and Dyer [35] by Cohen [36] were designed to extract all of the available... 

Biophysical characterization showed that a single HRP II protein bound 17 molecules of heme [35]. In an in vitro heme polymerization assay, HRP II promoted the synthesis of hemozoin, while controls, such as the proteins bovine serum albumin and lysozyme or the homopeptides polyhistidine, polylysine, and polyasparagine, did not. FT-IR analysis of the reaction product showed the characteristic vibrations of hemozoin. The polymerization activity had a pH maximum near 4.0, which dropped off precipitously near the pKa of histidine. The heme polymerization... 

Fig. (3). Hemozoin Production Mediated by Bionucleating Templates. Representative polymerization assay with 50 pM of hemin in 2 ml acetate buffer (500 mM, pH 4.8) at 37° C. BNTI and BNTII were used in 1 and 2 nmol amounts. Chloroquine (CQ, 100 pM) was included with BNT I and BNT n in inhibition reactions. Polyhistidine and bovine serum albumin in approximately 1 and 2 nmol amounts were used in protein control experiments. The blank control was the acetate buffer above. Base line amounts of insoluble aggregate are consistent with those previously reported under similar conditions.

As is often the case in multidisciplinary research, a number of different assays have been developed to monitor the inhibition of hemozoin aggregation (Table 1). Consequently, a great deal of confusion has arisen in the literature concerning the exact efficacy of a particular hemozoin inhibitor. For this reason, it is worthwhile to discuss the principal assays employed in the evaluation of antimalarial compounds and those assays specifically designed to evaluate heme aggregation inhibition. 

The possible mode of action of these drugs also emphasizes the dual importance of both the mechanism of transport, as well as hemozoin disruption. Goldberg et al. suggested that, in part, the molecular conformation of the metal complex may be important to pharmocological... 

In related work, Martiney et al. showed that metalloporphyrins with zinc or tin were capable of inhibiting hemozoin formation in trophozoite extracts [105]. Zn(II) and Sn(IV) analogs of heme were selected for study as a result of their known competitive interactions with heme in a number of systems and their use as pharmacological photosensitizers. The order of efficacy for these metalloporphyrins was ZnDPIX>SnPPIX ZnPPIX. This order is consistent with the proposed... 

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