BOVINE WHEY FRACTIONATION BASED ON CATION-EXCHANGE CHROMATOGRAPHY

Bovine whey proteins have potential applications in veterinary medicin e, food industry and as supplements for cell culture media. A fraction ation scheme for the economically interesting proteins, such as IgG, l actoferrin and lactoperoxidase, based on cation exchangers was the goa l of our investigations. A chromatographic process was developed where alpha-lactalbumin passes through the column and separation of the des ired proteins is achieved. Four different cation-exchange media (S-Hyp erD-F, S-Sepharose FF, Fractogel EMD SO3- 650 (S) and Macro-Prep High S Support) were compared in regard to their dynamic binding capacity f or IgG and their different elution behaviours when sequential step gra dients with NaCl buffers were applied. Peak fractions were analyzed by size-exclusion chromatography and sodium dodecyl sulphate-polyacrylam ide gel electrophoresis. Lactoperoxidase activity was monitored by the oxidation of o-phenylenediamine. In order to explain the different re solution behaviours, isocratic suns with pure standards of whey protei ns were performed. The k' values were calculated and plotted against s alt concentration. Fractogel EMD had the highest binding capacity for IgG, 3.7 mg/ml gel at a linear flow-rate of 100 cm/h, but the resoluti on was low compared to that with the other three media. S-Hyper D and S-Sepharose FF showed lower capacities, 3.3 and 3.2 mg/ml gel, respect ively, but exhibited better protein resolution. These effects could be partially explained by the k' versus salt concentration plots. The bi nding capacity of Macro-Prep S was considerably lower compared to that of the other resins investigated because its selectivity for whey pro teins was completely different. S-Sepharose FF and S-Hyper D combine r elatively high dynamic capacity for IgG and good resolution. Compared to studies with standard proteins, such as 100 mg/ml bovine serum albu min for S-Hyper D, their binding capacities were very low. Even after removal of low-molecular-mass compounds, the capacity could not be imp roved significantly. The running conditions (low pH) were responsible for the low protein binding capacity, since low-molecular-mass compoun ds in the feed do not compete with the adsorption of whey protein. The dynamic capacity did not decrease to a large extent within the range of flow-rates (100-600 cm/h) investigated. The dynamic capacity of Hyp erD and Fractogel was at least five times higher when pure bovine IgG was used for determination. In conclusion, S-Sepharose FF, S-Hyper D-F and Fractogel EMD SO3- 650 (S) are considered as successful candidate s for the large-scale purification of bovine whey proteins. (C) 1998 E lsevier Science B.V.