Reversible and oriented immobilization of histidine-tagged protein on silica gel characterized by frontal analysis

Abstract

This approach utilized N,N′-bis(carboxymethyl)-L-lysine (ANTA) coordinated to bivalent metal cation Ni²⁺, leaving free coordination sites for the reversible binding of gene recombinant histidine-tagged β₂-adrenoceptor onto macropore silica. The amount of transient metal nickel ion on the support was determined by atomic absorption spectrophotometry. The novel protein oriented immobilization β₂-AR column was evaluated by five β₂-adrenoceptor agonists, applying frontal analysis. The association equilibrium constant for ligands on the column was 1.98 × 10⁴ M⁻¹ for salbutamol, 3.43 × 10⁴ M⁻¹ for clenbuterol, 2.09 × 10⁴ M⁻¹ for tulobuterol, 1.84 × 10⁴ M⁻¹ for terbutaline, 1.71 × 10⁴ M⁻¹ for methoxyphenamine and corresponding concentrations at binding sites were 7.46 × 10⁻⁶ M, 1.82 × 10⁻⁵ M, 2.16 × 10⁻⁵ M, 8.29 × 10⁻⁶ M and 3.88 × 10⁻⁵ M, respectively. The results obtained from breakthrough and nonlinear fitting indicated that all the drugs have a single binding site on the β₂-adrenoceptor column. The present combined histidine-tagged protein method was reliable and exact in revealing interactions between receptor and drugs.