Binding of Lipophilic Nutrients to β-Lactoglobulin Prepared by Bioselective Adsorption

Abstract

The binding of the lipophilic nutrients, retinal, vitamin D2, and retinyl palmitate by beta-lactoglobulin was measured by analysis of changes in the fluorescence of the tryptophanyl residue of beta-lactoglobulin or the retinyl moiety. The fluorescence intensity of the tryptophanyl residue was quenched by retinoid or vitamin D binding but was enhanced by palmitate binding. The analysis of competitive binding experiments with palmitate indicated that retinal and palmitate did not compete for the same site; however, vitamin D2, which binds with a stoichiometry of 2, appeared to displace palmitate at higher concentrations. Also, the retinoids and vitamin D2 were bound more tightly than was palmitate. The results are consistent with the model in which the retinoids and vitamin D2 bind in the calyx formed by the beta-barrel; palmitate and a second molecule of vitamin D2 bind in a surface pocket near the dimer contact region. Retinyl palmitate, which has both moieties, appeared to bind at both sites.

Exploring the heat-induced structural changes of β-lactoglobulin -linoleic acid complex by fluorescence spectroscopy and molecular modeling techniques

“…Three independent tests were carried out in each case and SD was lower than 3.5 %. Inset: The emission spectrum of un-treated β-Lg (grey) and β-Lg-LA complex (black) (Frapin et al 1993;Wang et al 1998Wang et al , 1999. These studies also report that the β-Lg fluorescence intensity increased when fatty acids bound to the superficial pocket.…”

“…2b) , who quenched its fluorescence. Two lipophilic ligand binding sites have been reported for β-Lg: a central hydrophobic β-barrel and a superficial pocket (Wang et al 1998(Wang et al , 1999. It seems that fatty acids bind at the superficial pocket, while other molecules bind to the calyx Fig.…”

Rational Design for Crystallization of β-Lactoglobulin and Vitamin D3 Complex: Revealing a Secondary Binding Site

“…Although a secondary vitamin D binding site of LG has been proposed from some physicochemical experiments, [23][24][25]35,36 its existence and location have remained elusive and controversial. Several studies have clearly demonstrated that the binding stoichiometry between retinol or palmitic acid and LG is 1 where the central calyx of LG is responsible for retinol and palmitic acid binding, [21][22][23] but whether the binding of vitamin D to LG is 1 or 2 remains uncertain.…”

“…Wang et al proposed that LG possesses two potential binding sites for vitamin D: one is in the calyx formed by a -barrel and the other is near an external hydrophobic pocket between the R-helix and the -barrel. 24,25 In the previous study, we first showed that the relative binding of vitamin D to LG was 56% of the maximal binding at a vitamin D 3 /LG ratio of 1:1 and the binding stoichiometry of vitamin D 3 to LG was 2:1 relative to that 1:1 of retinol or palmitic acid verified using extrinsic fluorescence emission, fluorescence enhancement and quenching methods (Table 1). 26 Our previous data tended to support the view that LG comprises two vitamin D binding sites.…”

“…[19][20][21][22] The location of vitamin D binding sites has been controversial, yet most evidence points toward the calyx. It has been postulated that another site exists, [23][24][25] but it could not be verified by the crystal structure using the vitamin D 2 -LG complex. 10 In a previous study, we first conducted a ligand binding assay using the fluorescence changes by retinol, palmitic LG was 2:1, whereas that of retinol or palmitic acid was 1:1 (Table 1), 26 suggesting that there was another binding site for the vitamin D 3 molecule.…”