Characterization of human creatine kinase BB and MB isoforms by means of isoelectric focusing

Abstract

Isoforms of creatine kinase (creatine- N-phosphotransferase, CK, EC 2.7.3.2), BB and MB, were isolated from healthy human brain tissue and cardiac muscle, respectively, and were characterized by means of isoelectric focusing (IEF). CK-BB isoforms in Tris-HCl buffer were focused at p I 4.5 (a tissue form) and those in fresh sera from healthy adults were focused at p I 5.0, 5.1 and 5.2 (plasma forms). The IEF patterns of CK-BB isoforms were not altered following treatment with carboxypeptidase B and incubation in fresh serum at 37°C; thus, it was found that there was no lysine at the C-terminal of the CK-B subunit and CK-BB isoforms were not results of the removal of lysine. Three CK-BB isoforms in fresh sera were identified to be oxidized, intermediate and reduced forms from the anodal side, respectively, by treatment with hydrogen peroxide and 2-mercaptoethanol. The oxidized form of CK-BB seemed to have higher affinity to IgG than the other two plasma forms of CK-BB. On the other hand, CK-MB isoforms in Tris-HCl buffer were focused at p I 5.4 (a tissue form) with a minor band at p I 5.2 and those in sera were focused at p I 5.0, 5.1, 5.2 (plasma forms) and 5.4. Four CK-MB isoforms were identified to be reduced, intermediate and oxidized forms without lysine from the anodal side, respectively, and the cathodal band was a tissue form with lysine.