PROTEOLYSIS OF HUMAN CERULOPLASMIN - SOME PEPTIDE-BONDS ARE PARTICULARLY SUSCEPTIBLE TO PROTEOLYTIC ATTACK

Highly purified human ceruloplasmin [EC 1.16.31.1] was isolated from fresh donor blood in the presence of inhibitors of proteolysis and from stored retroplacental blood serum with and without inhibitors of proteolysis. According to the data of electrophoresis, ultracentrifuge sedimentation velocity and sedimentation equilibrium, all the ceruloplasmin samples were homogeneous within MW of 130,000. The dissociation of the samples treated by dodecyl sulfate, guanidine .cntdot. HCl and urea was studied by means of quantitative analytical and preparative electrophoresis and sedimentation equilibrium. The dissociation patterns depended on whether inhibitors were used in the isolation procedure. Polypeptides with MW of 130,000, 112,000 and 16,000 (minor component) were obtained if phenylmethylsulfonyl fluoride and/or 6-aminohexanoic acid were used; if these compounds were not used, the dissociation yielded additional polypeptides of MW of 100,000 (minor component), 64,000 and 48,000. Under proteolysis-favoring conditions the relative amount of these polypeptides increased. Prolonged storage of samples under sterile conditions without inhibitors of proteolysis resulted in a decrease of the relative amount of polypeptides with MW of 130,000, 112,000, 100,000, 64,000 and 48,000, contrary to that of the 16,000 MW polypeptide, which increased. At the same time new polypeptides appeared with MW of 42,000 and 21,500-23,000. Spontaneous specific fragmentation of the ceruloplasmin molecule is due to trace amounts of proteases, which seem to originate from blood plasma. Limited tryptic hydrolysis of the ceruloplasmin globule resulted in the appearance of polypeptides with the same MW observed in spontaneous fragmentation. The ceruloplasmin molecule in vivo is probably single polypeptide chain with at least 5 bonds which in vitro are the points of specific proteolytic fragmentation, yielding 6 principal fragments.