Human plasma lecithin:cholesterol acyltransferase. Preparation and use of immobilized p-aminophenylarsenoxide as a catalytic site-directed covalent ligand in enzyme purification.
A method is described for the preparation of p-aminophenylarsenoxide-linked carboxymethyl (CM) Bio-Gel A and its use as a specific, catalytic site-directed affinity chromatography ligand in the final stages of the purification of human plasma lecithin:cholesterol acyltransferase (LCAT) (EC 2.3.1.43). Previous mechanistic studies by us demonstrated that phenylarsenoxide derivatives, which are highly specific for vicinal thiols, could inhibit LCAT via a covalent interaction with the sulphydryl groups of the two catalytic cysteine residues and that this inhibition could be rapidly and completely reversed upon addition of 2,3-dimercaptopropanesulphonic acid. The ligand was covalently linked to CM Bio-Gel A activated through an N-hydroxysuccinyl ester formed by N-hydroxysuccinimide and dicyclohexylcarbodiimide in dry dimethyl sulphoxide; 87% of the added p-aminophenylarsenoxide was coupled to the CM Bio-Gel A in 3 h at 25 degrees C giving 2.3 mg of p-aminophenylarsenoxide per ml of gel. Homogeneous LCAT free of apo A-I, apo E, apo D and albumin was obtained in an 11% yield and purified 15,013-fold overall. A 13-fold purification was obtained by chromatography upon p-aminophenylarsenoxide-CM Bio-Gel A. This method is a useful final step in LCAT purification and will prove valuable in the purification of other proteins and compounds containing vicinal thiols.
Zhou
G Y
GY
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Jauhiainen
M
M
Stevenson
K
K
Dolphin
P J
PJ
D001152:Arsenicals; D002846:Chromatography, Affinity / Q000379:methods*; D005782:Gels; D006801:Humans; D008024:Ligands; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000097:blood* / Q000302:isolation & purification
D001152:Arsenicals; D005782:Gels; D008024:Ligands; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; C032642:4-aminophenylarsenoxide