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

¹ Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695-7624

The possibility of using biorecognition as an indicator of native structure in ß-lactoglobulin was investigated with an immobilized analog of retinal. Trans-retinal was covalently bound to 200-nm 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 ß-lactoglobulin in solution. Thus, the nonpolar portion of the ligand is largely responsible for binding as previously suggested. Zonal chromatography of ß-lactoglobulin and ß-lactalbumin mixtures or whey samples resulted in rapid separation of native ß-lactoglobulin 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 ß-lactoglobulin by chromatography were compared to literature values obtained from solubility measurements. The two methods are in excellent agreement.

Key Words: ßbeta;-lactoglobulin denaturation • immobilized trans-retinal • chromatography

Accepted on March 14, 1990