doi:10.1016/S0166-6851(97)00161-8
Copyright © 1997 Elsevier Science B.V. All rights reserved
Synthetic peptides corresponding to a repetitive sequence of malarial histidine rich protein bind haem and inhibit haemozoin formation in vitro
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Amit V. Pandey1, a, Ratanmani Joshib, Babu L. Tekwania, *, Ram L. Singhc and Virender S. Chauhanb
a Division of Biochemistry, Central Drug Research Institute, Lucknow 226001, India
b Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Road, New Delhi 110 067, India
c Department of Biochemistry, R.M.L. Avadh University, Faizabad 224 001, India
Received 17 February 1997;
revised 24 June 1997;
accepted 10 September 1997.
Available online 29 January 1998.
Abstract
Synthetic peptides containing a repetitive hexapeptide sequence (Ala-His-His-Ala-Ala-Asp) of malarial histidine-rich protein II were evaluated for binding with haem in vitro. The pattern of haem binding suggested that each repeat unit of this sequence provides one binding site for haem. Chloroquine inhibited the haem–peptide complex formation with preferential formation of a haem–chloroquine complex. In vitro studies on haem polymerisation showed that none of the peptides could initiate haemozoin formation. However, they could inhibit haemozoin formation promoted by a malarial parasite extract, possibly by competitively binding free haem. These results indicate this hexapeptide sequence represents the haem binding site of the malarial histidine-rich protein and possibly the site of nucleation for haem polymerisation.
Author Keywords: Malaria; Haemoglobin; Haem; Haemozoin; Histidine-rich protein; Chloroquine
Fig. 1. Spectra of haem–peptide complex. Concentration of peptide HRP IIC was 20 μM and haem 10 μM. An aliquot of 10 μM haem was added in the reference.
Fig. 2. Interaction of haem with synthetic HRP peptides. (A) Binding of haem to HRP IIC. Each point represents the mean±SD of triplicate observations. HRP IIC (20 nmol) was titrated against haem in increments of 5 nmol. Total increase in the volume during experiment was less than 5%. (B) Titration of different peptides with haem. The number of hexamer units in the peptides is plotted against the number of haem bound per mole of peptide.
Fig. 3. Effect of peptides and haemozoin on haem polymerisation. Different components were added as displayed in the table below the bar diagram. Concentrations of components was as follows: haem, 100 μM; peptide, 100 μM; purified haemozoin, 5 nmol (equivalent of haem); parasite extract containing 2.5 nmol of haemozoin. Other conditions were as described in Section 2. Results are displayed as mean±SD of triplicate experiments.
Fig. 4. Inhibition of Plasmodium yoelii haem polymerisation activity by HRP II peptides. Each bar represents mean±SD of triplicate experiments. Haemozoin formed in the control during reaction was 4.5 nmol.
Fig. 5. Interaction of chloroquine with haem–peptide complex formation. Lanes: 1, haem–peptide complex; 2, haem–peptide interaction in presence of chloroquine; 3, haem–chloroquine complex; 4, haem. Concentrations of components were as follows: peptide HRP IIC, 40 μM; haem, 20 μM; chloroquine, 40 μM. O, origin; SF, solvent front.
1Present address: Malaria Group, ICGEB, Aruna Asaf Ali Road, New Delhi 110 067, India.
*Corresponding author. Fax: +91 522 223405; e-mail: root@cscdri.ren.nic.in
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