Biotin-binding protein from egg yolk. A protein distinct from egg white avidin.

Biotin-binding protein has been purified 2000-fold from chicken egg yolk with a 1 to 2% yield. The puriflcation includes l-butanol extraction of yolk lipids, phosphocellulose chromatography of water-soluble proteins, affinity binding and elution from biotinylSepharose, and gel filtration chromatography. Since the protein biotin-binding sites are saturated with very tightly bound biotin, a novel procedure was developed to displace bound biotin with biotinyl-sepharose. This exchange occurs with continuous diafdtration at 66°C in a pressure concentrator. All of the biotin-binding protein can be bound to the amty matrixinlOto16h. The purified protein is a tetramer, contains carbohydrate, and is homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the native tetramer is about 74,300. A single NH%-terminal valine is found, suggesting that the four subunits are identical. The specific activity of 12 to 13 pg of biotin/mg of protein indicates that each subunit binds 1 molecule of biotin. The isoelectric pH is about 4.6. The approximately 161 amino acids per subunit account for slightly more than 88% of the subunit molecular weight of 19,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In comparison with avidin, the biotin-binding protein from egg white, egg yolk biotin-binding protein appears to be distinct in terms of p1 (pH 4.6 as opposed to pH lo), amino acid composition, NH&erminal amino acids (valine as opposed to alanine), and cyanogen bromide cleavage pattern. Antibodies directed against either protein will not cross-react with the other protein. The rate of release of biotin is more than l,OOOfold faster from the yolk protein at all temperatures tested. The proteins are proposed to arise from separate genes.