CHARACTERIZATION OF THE COMPLEX OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 WITH TISSUE-TYPE PLASMINOGEN-ACTIVATOR BY MASS-SPECTROMETRY AND SIZE-EXCLUSION CHROMATOGRAPHY

Glycosylated human plasminogen activator inhibitor type 1 (PAI-1), pro duced in Chinese hamster ovary (CHO) cells, showed a variety of compou nds with different molecular weights when subjected to electrospray ma ss spectrometry (ES-MS), owing to the heterogeneity of the carbohydrat e chains. However, non-glycosylated human PAI-1, produced in E. coli, gave rise to a prominent species with a molecular weight of 42774, con sistent with the amino-acid sequence. A non-glycosylated mutant of the proteinase domain (B-chain) of tissue-type plasminogen activator (tPA ) produced in C 127 cells, had a molecular weight of 28 168. Full-leng th, glycosylated, tPA showed a large heterogeneity in molecular mass. For a mass study, a tPA-PAI-1 complex was formed, composed of non-glyc osylated PAI-1 and non-glycosylated B-chain, This complex was remarkab ly stable at room temperature in buffer with a neutral pH. The mass sp ectrum of the complex provided two main species, a peptide with a mass of 3803 and a dominating species of 67 133. These masses are consiste nt with a complex where PAI-1 is cleaved at the P1-P1' position. A tra ce of a species with a molecular mass of 70 942 was also found, corres ponding to the complete, non-dissociated complex with PAI-1. Separatio n of the cleaved peptide, corresponding to the hydrophobic C-terminal 33 amino-acid residues of PAI-1, from the complex, was achieved by siz e-exclusion chromatography in the presence of 30% acetonitrile. Thus, in the complex between tPA and PAI-I, the proteins are held together b y a tight covalent bond, but the C-terminal cleaved peptide of PAI-1 i s only bound to the complex by hydrophobic forces. To assess whether t his is specific to the tPA B-chain alone, experiments with the complex of full-length, glycosylated tPA and glycosylated PAI-1 were also per formed, and it was possible to demonstrate the release of the C-termin al PAI-1 peptide by chromatography, mass spectrometry, as well as by S DS-PAGE.