Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography

Human plasma paraoxonase (PON1) is calcium-dependent enzyme that hydrolyses esters, including organophosphates and lactones and exhibits anti-atherogenic properties. Human phosphate binding protein (HPBP) was discovered as contaminant during crystallization trials of PON1. This observation and uncertainties for the real activities of PON1 led us to re-evaluate the purity of PON1 preparations. We developed a hydroxyapatite chromatography for the separation of both HDL-associated proteins. We confirmed that: (1) HPBP is strongly associated to PON1 in HDL, and generally both proteins are co-purified; (2) standard purification protocols of PON1 lead to impure enzyme; (3) hydroxyapatite chromatography allows the simultaneous purification of PON1 and HPBP. (c) 2006 Elsevier B.V. All rights reserved.