Analysis of Ligand Binding and β-Lactoglobulin Denaturation by Chromatography on Immobilized Trans-Retinal

The possibility of using biorecognition as an indicator of native structure in plactoglobulin was investigated with an nal was covalently bound to 2Wnm pore diameter aminopropyl-controlled-pore glass by reductive amination with sodium cyanoborohydride. The immobilized trans-retinal was packed into a 3-mm x 150-mm glass column and incorporated into an HPLC unit. Using continuous elution analytical affinity chromatography, the measured dissociation constant is 35.6 nM, which is similar to the value reported for binding of retinol to plactoglobulin in solution. Thus, the nonpolar portion of the ligand is largely responsible for binding as previously suggested. Zonal chromatography of p-lactoglobulin and a-lactalbumin mixtures or whey samples resulted in rapid separation of native plactoglobulin from the denatured form and all other proteins. Samples containing varying quantities of native and denatured protein were prepared by heat treatment. Analyses of these samples for the amount of native plactoglobulin by chromatography were compared to literature values obtained from solubility measmments.