<b>CHARACTERIZATION OF HEMOZOIN FROM LIVER AND SPLEEN OF MICE INFECTED WITH P<i>LASMODIUM YOELII</i>, A RODENT MALARIA </b><b>PARASITE </b>

Abstract

A non-enzymatic protocol for the purification of hemozoin, the malaria pigment, accumulated in the liver and spleen of mice infected with Plasmodium yoefii has been developed. The procedure, including repeated extraction with 2.5% SDS followed by washings with methanol, chloroform and bicarbonate buffer, completely removes the non-hemozoin heme and other non-covalently adhered biomolecules. The purified hemozoin contains 94-98% ferriprotoporphyrin IX as quantitated by spectral analysis. A parasitemia-dependent accumulation of hemozoin was demonstrated which was more marked in the liver than in the spleen.

Formation of haemozoin/β‐haematin under physiological conditions is not spontaneous

“…However, overnight incubation of haematin at 80°C yielded a product which was not soluble in bicarbonate buffer and 2.5% SDS. The extracts of Plasmodium yoelii, Plasmodium berghei and Plasmodium knowlesi also contain a bicarbonate buffer/SDS insoluble component [16]. Incubation of haematin in 4.5 M acetate at 80°C for 12 h yielded two components on TLC analysis (Table 2) one corresponding to haem and another corresponding to [3-haematin.…”

“…Malarial haemozoin was purified from the erythrocytes highly infected with Plasmodium yoelii (parasitemia > 60%) or liver from the infected animals, according to the method described earlier [16]. Nonbiological/chemical synthesis of [3-haematin was carried out by incubating haematin in 4.5 M sodium acetate overnight at 80°C [13].…”

“…Nonbiological/chemical synthesis of [3-haematin was carried out by incubating haematin in 4.5 M sodium acetate overnight at 80°C [13]. Purity and polymeric nature of malarial haemozoin and [3-haematin were ascertained by the analysis of elemental composition, visible spectra, differential solubility characteristics and also by infrared spectroscopy [16]. To check the formation of [3-haematin under physiological conditions non-biologically haematin (5.0 mg/ml) was incubated in solutions with various concentrations of sodium acetate (pH 4.5) as described by Egan et al [14].…”

“…The haem-acetate incubation mixtures were filtered over nitrocellulose filters (0.45 ~m, Millipore, USA) as described by Egan et al [14] and subjected to different kinds of washings. The amount of ferriprotoporphyrin IX (1996) [189][190][191][192] (haem) retained on the filters after the washing was measured spectrophotometrically [16] after submerging the filters in 0.1 N NaOH for overnight. For recording infrared spectra the solid malarial haemozoin/~-haematin/haematin/haem-acetate incubation products were dried over silica gel and phosphorous pentaoxide and IR spectra were recorded on a Perkin-Elmer 881 (Fig.…”

“…However, the conditions required for this are highly non-physiological. The most important criterion for the separation of haemozoin/[3-haematin from the haematin/haem is their differential solubilities in alkaline bicarbonate buffer (pH 9.0 9.5) and 2.5% sodium dodecyl sulfate (SDS) [13,15,16]. Haem/haematin is readily soluble in these solvents while haemozoin/13-haematin are insoluble.…”