High-performance liquid chromatographic analysis of biological and chemical heme polymerization.
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.
Berger
B J
BJ
Picower Institute for Medical Research, Manhasset, New York 11030, USA.
Bendrat
K
K
Cerami
A
A
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006418:Heme / Q000737:chemistry / Q000302:isolation & purification*; D006427:Hemin / Q000737:chemistry*; D010963:Plasmodium falciparum / Q000737:chemistry*; D011108:Polymers / Q000302:isolation & purification
D011108:Polymers; D006418:Heme; D006427:Hemin