Isolation, characterization, and comparison of the solubilized particulate and soluble folate binding proteins from human milk.

Human milk contains both a particulate and a soluble folate binding protein. Following solubilization of the particulate folate binding protein with 1%Triton X-100, the particulate and soluble folate binding proteins were purified 13,000-fold using pteroylglutamic acid-Sepharose as the major purification step. Each of the purified proteins gave a single band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The soluble folate binding protein had a molecular weight of approximately 40,000 by either sodium dodecyl sulfate-polyacrylamide gei electrophoresis or gel filtration while the solubilized particulate folate binding protein had a molecular weight of 45,000 by sodium dodecyl sulfatepolyacrylamide gel electrophoresis following removal of Triton X-100 and 160,000 by gel filtration in 1% Triton X-100. The discrepancy in the molecular weight of the particulate folate binding protein in the presence and absence of Triton X-100 was shown to be due to Triton X-100 micelle binding. Minimal to no detergent binding was demonstrable for the soluble folate binding protein or the recently purified human placental folate receptor (Antony, A. C., Utley, C. S., Van Horne, K. C., and Kolhouse, J. F. (1981) J. Biol. Chem 256, 96849692). Amino acid and carbohydrate analyses indicated that the particulate folate binding protein contained 39,000 g of amino acid and 4,800 g of carbohydrate per mol of bound folate, while the soluble folate binding protein contained 21,000 g of amino acid and 6,000 g of carbohydrate per mol of bound folate. Both proteins had similar stoichiometry of binding with 1 mol of folate bound per mol of protein, identical and higher affinities for pteroylglutamic acid than stable folate derivatives, and shared antigenic determinants indicated by single precipitin lines of identity on immunodiffusion with chicken antisera raised against either purified protein.