Comparative studies on two active enzyme forms of human urinary urokinase. I. Purification by serial column chromatography and homogeneity analyses of molecular weight and isoelectric point.

Abstract

A method for the efficient further purification of 2 active forms of urokinase (UK) [EC. 3.4.21.31] from partially purified human urinary UK was established by serial column chromatography on Sephadex G-100, Sephadex G-75 and SP-Sephadex C-50. According to Ferguson plots 2 active forms showed MW of 5.5 .times. 104 (H-UK) and 3.6 .times. 104 (L-UK). Both UK forms were homogeneous by SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis, gel gradient electrophoresis and Ouchterlony''s double immunodiffusion methods. Amino acid analysis showed that there was no marked difference in composition between H- and L-UK. The specific activity of H-UK was 1.2 .times. 105 IU/mg protein, which exceeds the highest specific activity that was ever obtained by other investigators, and that of L-UK was 1.52 .times. 105 IU/mg protein. This serial column chromatography procedure is apparently effective for the purification of H-UK, which is known to be a pharmacologically advantageous form. As judged by isotachoelectrophoresis, H-UK contained 5 subforms with pI [isoelectric point] values of 8.7, 8.9, 9.1, 9.2 and 9.4, and L-UK contained the same number of subforms with pI values of 7.5, 8.3, 8.8, 9.4 and 9.7. The pI values of the subforms with the highest specific activity were 9.4 for H-UK and 8.3 and 8.8 for L-UK. Judging from the dissimilarity in the pI values of UK preparations currently obtained, the subforms of UK obtained after purification may not correspond to the forms present in vivo, but may be artifacts of the purification procedures used.