1982 Volume 23 Issue 4 Pages 319-324_1
The water-soluble fraction of soybeans was extracted with 0.01M Tris-hydrochloric acid buffer (pH 7.4). The high molecular weight fraction (F-I, MW.>10, 000) was separated from the soluble fraction by gel filtration on Sephadex G-50. The chemical forms of copper (Cu) in F-I were investigated.
F-I was separated into five components by chromatography on a Sephadex G-100 column (2.0×70cm). Most of the endogeneous copper in F-I was eluted in the highest molecular weight fraction (F-IA, MW.>100, 000), the major component in F-I. When copper was added exogeneously to F-I, further copper could bind with F-I and over half the bound copper was found in F-IA.
Copper-binding affinities of F-I and F-IA were determined by equilibrium dialyses. The dissociation constants of F-I and F-IA for copper were calculated to be 2.3×10-5M and 2.0×10-5M, respectively, by fitting the Scatchard equation to the copper binding equilibrium curves. Maximal binding amounts of copper with F-I and F-IA were estimated to be 6, 000 and 5, 700ppm, respectively.
Artificial [F-I]-Cu complex containing a high level copper was prepared by the addition of copper to F-I. The copper content of the artificial complex was almost constant (2, 700ppm) in several experiments. The stability of this complex were studied by equilibrium dialyses against EDTA-2Na, 2-mercaptoethanol (2-ME), organic acids (6 kinds), amino acids (15 kinds) or metal ions (10 kinds). The artificial complex was unstable to EDTA-2Na, histidine, Zn2+, Hg2+ or Cd2+. On the other hand, the complex was found to be stabilized by compounds having SH groups: 2-ME, cysteine and glutathione (reduced form).
