Gel Filtration of Copper Binding Components in Soybeans

Abstract

Copper binding components in soybeans were extracted with buffer solution and separated by gel filtration.1. 60% of Cu in soybeans was extracted with 0.01M Tris-hydrochloric acid buffer solution (pH 7.4). The protein content of the crude extract was approximately 63%.2. The crude extract was characterized by gel filtration on Sephadex G-50. It was separated into two fractions (F-I, m. w.>ca. 10, 000 and F-II, m. w.<ca. 10, 000). F-I contained 20ppm Cu and 80-90% protein, while F-II contained 24ppm Cu and ca. 1% protein.3. F-I was treated with enzymes. No change was found in the gel filtration profile of F-I on Sephadex G-50 after treatment with trypsin or α-amylase.4. The binding capacity of F-I to Cu was also determined. When Cu was added in batches to F-I, the amount of bound Cu increased with the amount added. When 80μg of Cu was added to 20mg of F-I, the concentration of Cu in F-I increased over 80 times (1, 700ppm) from the original level (20ppm).5. Cu in F-II was eluted together with nitrogen-containing material, which gave a positive ninhydrin reaction. There may be an interaction between Cu and peptides in F-II.