High-performance liquid chromatographic analysis of biological and chemical heme polymerization

Anal Biochem. 1995 Oct 10;231(1):151-6. doi: 10.1006/abio.1995.1514.

Abstract

Free hematin can be converted to a stable polymer both chemically, by heating hematin in acid suspensions, or biologically, in the food vacuoles of malaria. A high-performance liquid chromatographic assay has been developed which can separate and quantitate both free hematin and the polymer (beta-hematin), based on the differential solubility of the two compounds. Ion-pair reverse-phase chromatography, utilizing tetramethylammonium chloride and heptane sulfonate as the ion-pair agents in the presence of 40% acetonitrile, was performed on a polymeric-resin-based column with a phenyl bonded phase. Initiating the runs at pH 2.5 led to elution only of the free hematin, and a subsequent shift to pH 12.0 converted the beta-hematin back to hematin which then eluted separately. The method was found to have a linear range of detection from 78 pmol to 20 nmol injected hematin and intra- and interday variations of 9.71 and 12.46%, respectively. The assay was used to study several basic aspects of heme polymerization in vitro, including effects of hematin and beta-hematin concentration on the rate of polymerization.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid / methods*
  • Heme / chemistry
  • Heme / isolation & purification*
  • Hemin / chemistry*
  • Plasmodium falciparum / chemistry*
  • Polymers / isolation & purification

Substances

  • Polymers
  • Heme
  • Hemin