title: Immobilization of alpha(1)-acid glycoprotein for chromatographic studies of drug-protein binding.
abstract: A new method for preparing immobilized alpha1-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4 degrees C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37 degrees C were 2.7 x 10(6) and 4.2 x 10(6) M(-1), respectively, with linear van't Hoff plots being obtained between 5 and 37 degrees C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors.
authors.affiliation: Chemistry Department, University of Nebraska, Lincoln, NE 68588-0304, USA.
authors.forename: Hai
authors.initials: H
authors.lastname: Xuan
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004353:Drug Evaluation, Preclinical; D006801:Humans; D009961:Orosomucoid / Q000737:chemistry* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D004364:Pharmaceutical Preparations / Q000737:chemistry; D011433:Propranolol / Q000737:chemistry; D011485:Protein Binding; D013237:Stereoisomerism; D013696:Temperature
chemical_list: D009961:Orosomucoid; D004364:Pharmaceutical Preparations; D011433:Propranolol