title: A new affinity gel for the purification of α-carbonic anhdrases.
abstract: The new affinity gel reported in this study was prepared using EUPERGIT C250L as a chromatographic bed material, to which etylenediamine spacer arms were attached to prevent steric hindrance between the matrix and ligand, and to facilitate effective binding of the CA-specific ligand, of the aromatic sulfonamide type for the purification of α-carbonic anhydrases (Cas; EC 4.2.1.1). Indeed, the aminoethyl moieties of the affinity gel were derivatized by reaction with 4-isothiocyanatobenzenesulfonamide, with the formation of a thiourea-based gel, having inhibitory effects against CAs. Both bovine erythrocyte carbonic anhydrase BCA and human (h) erythrocyte CA isoforms I, II (hCA I and II) have been purified from hemolysates, by using this affinity gel. The greatest purification fold and column yields for BCA and for cytosolic (hCA I + II) enzymes were of 181-fold (21.07%) and 184-fold (9.49%), respectively. Maximum binding was achieved at 15 °C and I = 0.3 ionic strength for α-carbonic anhydrases.
authors.affiliation: Department of Chemistry, Faculty of Science and Literature, Balikesir University , Balikesir , Turkey .
authors.forename: Aysegul
authors.initials: A
authors.lastname: Sahin
authors.forename: Semra
authors.initials: S
authors.lastname: Isık
authors.forename: Oktay
authors.initials: O
authors.lastname: Arslan
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.forename: Ozen Ozensoy
authors.initials: OO
authors.lastname: Guler
mesh_terms: D000818:Animals; D024401:Carbonic Anhydrase I / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology; D005029:Ethylenediamines / Q000737:chemistry*; D005782:Gels; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D013449:Sulfonamides / Q000737:chemistry*; D013890:Thiourea / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D005029:Ethylenediamines; D005782:Gels; D008024:Ligands; D013449:Sulfonamides; D024401:Carbonic Anhydrase I; D024402:Carbonic Anhydrase II; D013890:Thiourea
keywords: Affinity chromatography; carbonic anhydrase; purification