Purification of biotin-binding protein from chicken egg yolk and comparison with avidin

Abstract

A simple alternative procedure for the purification in higher yields of the biotin-binding protein from the chicken egg yolk in a ligand-free form is described. The isolated protein was homogenous by the criteria of polyacrylamide gel electrophoresis, gel filtration chromatography, immuno-double diffusion and immunoelectrophoresis. The protein had an M_r of 72000 ± 2000 and was a homotetramer of subunit M_r of 18000 ±1000. It bound [¹⁴C]biotin in the molar ratio of 1:4 with an association constant (K_a) of 0.58 10¹²M^-1. The yolk biotin-binding protein and avidin exhibited qaulitatively similar spectral changes on interaction with biotin and p-hydroxyazobenzoic acid, but quantitatively these changes were more pronounced with avidin. Despite the lack of gross immunological cross-reactivity between the two biotin-binders, evidence based on immunological techniques for some degree of common conformational characteristics restricted to or around the ligand-binding sites of the two proteins was adduced. The mixed subunits of the two proteins failed to form hetero-oligomers on reconstitution.