Mixtures of four proteins: sperm whale myoglobin, horse myoglobin, transferrin, albumin with isoelectric points (pI), 8.3, 6.8-7.8, 5.9, 4.7 respectively, were successfully separated by chromatofocusing in the column packed with PBE 94 under the pH gradient generated by the elution of Polybuffer.
In developing the pH gradient by the internal method, PBE 94 was superior to DEAE Bio-Gel A, and the shape of pH gradient depended on the concentration of equilibration buffer, column bed length, flow rate and the concentration of elution buffer. Using the buffering capacity values of the buffers and the ion exchangers, the model calculations of the pH gradients were done and consistent with the experimental results.
By the batch adsorption of each protein at 20℃, the partition coefficients of proteins were determined at several pH values near the isoelectric points, and a separation scheme was proposed.
In separating the proteins by chromatofocusing, the pH gradients and the conductivity curves of eluent showed severe discontinuous points at the first stage of elution due to the large tolerance of Donnan potential at the pH front, but later on the smooth pH gradient was sustained. Mixtures of four proteins were separated out in the order of pI values with 65-100% yields, and the deviations of the elution pH values of the proteins from their pI values were less than 0.25 pH unit.
