Characterization of Ileal Vitamin B₁₂ Binding Using Homogeneous Human and Hog Intrinsic Factors

Elucidation of the mechanism of intrinsic factor (IF)-mediated vitamin B₁₂ (B₁₂) binding to ileal binding sites has been hampered by the use of crude or only partially purified preparations of IF in previous studies. We have used homogeneous human IF and hog IF isolated by affinity chromatography to study [⁵⁷Co]B₁₂ binding to ileal mucosal homogenates. The following observations were made: (a) Human IF-B₁₂ and hog IF-B₁₂ were bound to human, monkey, hog, dog, rabbit, mouse, hamster, and guinea pig ileal, but not jejunal, homogenates in amounts significantly greater than free B₁₂ or B₁₂ bound to five other homogeneous B₁₂-binding proteins; (b) only IF-mediated B₁₂ binding was localized to ileal homogenates and was inhibited by EDTA; (c) values for the association constant (K_a) for the various ileal homogenates mentioned above and human IF-B₁₂ and hog IF-B₁₂ ranged from 0.3 × 10⁹ M^-1 to 13.0 × 10⁹ M^-1. Apparent differences in the K_a for human IF-B₁₂ and hog IF-B₁₂ existed in most species; (d) the number of ileal IF-B₁₂ binding sites per gram (wet weight) of ileal mucosa ranged from 0.3 × 10¹² to 4.9 × 10¹². The same value was always obtained with human IF-B₁₂ and hog IF-B₁₂ for any given homogenate preparation; (c) 100-fold excesses of free B₁₂ or human IF and hog IF devoid of B₁₂ did not significantly inhibit human IF-B₁₂ and hog IF-B₁₂ binding to human and hog ileal homogenates.

These experiments performed with homogeneous IF indicate that: (a) gastric factors other than IF are not required for B₁₂ binding to ileal IF-B₁₂-binding sites: (b) the mechanism of ileal IF-B₁₂ binding is different from that of free B₁₂ or of B₁₂ bound to non-IF-B₁₂-binding proteins; (c) human IF and hog IF have different structures; (d) human IF-B₁₂ and hog IF-B₁₂ bind to the same ileal binding sites; and (c) human and hog ileal IF-B₁₂ binding sites bind free B₁₂ and human and hog IF devoid of B₁₂ poorly, if at all.

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