FAST PROTEIN LIQUID-CHROMATOGRAPHY PURIFICATION OF HYDROPHOBIC FRACTION OF BOVINE-MILK PROTEASE-PEPTONE AND CHARACTERIZATION BY BIDIMENSIONAL ELECTROPHORESIS

Bovine milk hydrophobic fraction of proteose-peptone was prepared by hydrophobic interaction fast protein liquid chromatography. This method has several advantages such as high rapidity, simple good reproducibility and less denaturation. The proteose-peptone was eluted from a TSK-Phenyl-5PW column with a 1 M-0 M ionic strength gradient of NaH2PO4, pH 6.8, using a 6 ml/min flow rate for 56 min. The quantity of protein injected was 62.5 mg; however, it could be increased up to 100 mg. The elution order was beta-CN-4P < BSA (1.6 % of total N) < Beta-CN-5P < beta-CN-1P. The hydrophobic fraction was obtained in pure water at the end of the gradient (17.3 % of total N). A proteose-peptone cartograph was achieved by bidimensional electrophoresis. This hydrophobic fraction represented three principal zones of M(r) 30 000-28 000, 19 000 and 11 000, which were respectively composed of 13, 4 and 2 principal spots distributed between 4.9 and 6.1 isoelectric points (IP). These spots corresponded to glycoproteins. Beta-CN-5P gave three spots of 4.7, 5.0 and 5.1 IP which migrated to M(r) 18 000 while beta-CN-1P was identified as M(r) 9000 in two spots of 5.1 and 5.3 IP.