Folate Binding Protein in Bovine Milk Occurrence and Properties Studied with Surface Plasmon Resonance

Surface plasmon resonance (SPR) based biosensors generate real-time binding data and is therefore well suited to analyse binding kinetics of two interacting molecules as well as for concentration measurements. Folate-binding protein (FBP) is present in, among others, milk and may affect intestinal absorption of folate. The objectives of this work were to investigate both the occurrence of bovine FBP in milk and FBP ligand binding properties using the SPR technique. In order to understand and predict the impact of FBP on folate stability and absorption of native or added folates (fortification) an SPR assay that detects both the free (unliganded) and total FBP content in milk was developed. The assay showed good compliance with an existing ELISA method that measures the total FBP content. It was shown that approx. 50% of the total FBP content in milk is not bound to folate. The SPR assay was further used for screening of the occurrence of free and total FBP in milk from Swedish diary cattle. A large variation in FBP levels among cows was observed. The binding kinetics between FBP and pure stereoisormeric forms of various folate derivatives were investigated. The results demonstrated that the interactions between FBP and the major forms of folate derivatives differ considerably. Each of the binding responses shown was well described by a 1:1 interaction model. The two folate forms that do not occur in nature, that is, folic acid and [6R]-5-CH3H4folate, had the highest affinity for FBP, 20 and 160 pmol/L, respectively. The influence of different folate derivatives on the denaturation temperature of FBP and effect of pasteurization and UHT treatment on FBP binding to folate were studied. The binding of folate increased the denaturation temperature of FBP. The ability of FPB to bind folate was eliminated by UHT treatment but not by pasteurization, regardless of folate form