Purification of Human Serum Paraoxonase-1

Membrane proteins play key roles in fundamental biological processes, such as transport of molecules, signalling, energy utilization, and maintenance of cell and tissue structures. Serum paraoxonase (PON1, EC 3.1.8.1.) is a protein of 354 amino acids with a molecular mass of 43 kDa is a high density lipid (HDL) associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. It is highly conserved in mammals but is absent in fish, birds, and invertebrates, such as arthropods. Purification of PON1 has been challenging for a long time. Here, we report a novel purification technique for this enzyme, which allowed us to obtain human serum paraoxonase 1 (hPON1) using straightforward chromatographic methods. The PON1-specific inhibitor 2-hydroxyquinoline almost completely inhibited paraoxonase and lactonase activities, while only moderately inhibiting arylesterase activity. By this method the purity of the isolated PON-1 was increased many folds. Later the purified sample was assessed for its paraoxonase, lactonase and arylesterase activities.