Distribution of Zinc and Purification of Copper Complex in the Water-soluble Low-molecular-weight Fraction of Soybeans

Abstract

The water-soluble fraction of soybean was extracted with 0.01M Tris-hydrochloric acid buffer (pH 7.4). The low-molecular-weight fraction (F-II, MW<10, 000) was separated from the soluble fraction by gel filtration on Sephadex G-50. The distribution of zinc (Zn) and the chemical form of copper (Cu) were studied.The concentrations of Cu and Zn in the water-soluble fraction were 26.6 and 107ppm, respectively. Zn in the water-soluble fraction was observed only as the bound form. Zn in F-II was always eluted together with Cu in F-IIB by gel filtration on both Sephadex G-25 and G-10. The binding affinity of Zn in F-IIB was found to be less than that of Cu. When Cu was added exogenously to the water-soluble fraction, Zn in F-IIB shifted to F-I. However, the addition of Zn did not cause a shift of Cu in F-IIB.The purification of the Cu complex in F-IIB was carried out by the application of anion exchange column chromatography (AG 1×2, DEAE-Sephadex A-25). Cu and Zn in F-IIB were held on the AG 1×2 column and both were eluted with 0.01M acetic acid. The dissociation of Zn from the ligands was observed in the chromatography, but Cu was always eluted together with nitrogen-containing materials which were ninhydrin-positive. The Cu complex in F-IIB gave a blue band on the DEAE-Sephadex A-25 column, but after purification, it did not have any absorption spectrum in the visible region. The Cu complex had the same elution point on Sephadex G-10 before and after the purification.The nitrogen/Cu ratio in the partially purified Cu complex was approximately 4-6 mole/mole. The UV spectrum of the partially purified Cu complex showed strong bands at 250nm and a shoulder at around 225nm. Amino acid analysis gave several major peaks (Asp, Glu, Thr, Ser, Gly, Cys and 1-MeHis). The partially purified Cu complex showed a characteristic ESR spectrum of Cu (II) complexes.