INTERACTION OF SUCROSE ESTERS WITH SKIM MILK-PROTEINS AS CHARACTERIZED BY AFFINITY-CHROMATOGRAPHY

The interaction of sucrose ester, P-1670 (70% pure palmitic acid monoe ster), with milk proteins was examined by affinity chromatography. The ester was covalently bound to 3-aminopropyl controlled-pore glass by periodate oxidation of the carbohydrate to aldehyde, formation of the Schiff base, followed by reduction to secondary amine. In phosphate bu ffer, pH 7, alpha-LA did not appear to interact with sucrose ester, bu t beta-LG exhibited binding. However, casein micelles were very tightl y bound, and caseins could only be eluted following addition of urea. On application of skim milk, the strengths of the interactions, as ref lected by their elution volumes, decreased in the order: alpha(s)-CN, kappa-CN, beta-CN, beta-LG, and alpha-LA. Micelles dissociate after ad dition of urea, allowing individual caseins to interact with sucrose e ster moieties in the presence of increasing urea concentrations. Appar ent dissociation constant was 2.04 mu M for the binary complex using s olutions of pure beta-LG in agreement with the value for soluble compl exes measured by other methods. Hydrophobic interaction appears to be the major force stabilizing complex formation, even though the affinit ies of the individual caseins do not follow the order of their overall hydrophobicities. Tight binding of micelles to the immobilized sucros e esters suggests that the alkyl chains are able to insert into hydrop hobic regions of surface submicelles.