DYNAMIC STRUCTURAL AND FUNCTIONAL-RELATIONSHIPS IN RECOMBINANT PLASMINOGEN-ACTIVATOR INHIBITOR-1 (RPAI-1)

The conformational characteristics of active, latent, and denatured re combinant plasminogen activator inhibitor-1 (rPAI-1) were compared usi ng UV spectroscopy, spectrofluorimetry and circular dichroism (CD) tec hniques. The UV absorbance wavelength maxima in all preparations appro ximated 280 nm, while the extinction coefficients of active and latent rPAI-1 differed by up to 60%. When incubated at 37-degrees-C, the A28 0 of latent rPAI-1 was quite stable while the A280 of active rPAI-1 sp ontaneously increased, eventually approximating that of latent rPAI-1. Alkali difference spectroscopy yielded markedly divergent titration p atterns for active and latent rPAI-1, suggesting that the tyrosine res idues present in active and latent rPAI-1 differ in terms of solvent e xposure. At an excitation wavelength of 280 nm, active rPAI-1 exhibite d the greatest relative fluorescence quantum yield. The relative fluor escence of latent and denatured rPAI-1 were less than that of active P AI-1, and the emission maxima of both species were slightly red-shifte d in comparison to that of active rPAI-1, suggesting that at least one of the four tryptophan residues present in rPAI-1 is less exposed to the aqueous environment in the active form of the molecule. In contras t, the derived secondary structures based on CD of active and latent r PAI-1 were nearly identical, with both moieties exhibiting approx. 40% alpha-helix and 15% beta-sheet. Taken together, these spectroscopic d ata provide evidence supporting the hypothesis that active and latent PAI-1 differ in terms of their tertiary conformation and aromatic resi due exposure, while their secondary structures appear generally compar able. Furthermore, denaturant-induced reactivation of latent rPAI-1 pr oduces a partially active rPAI-1 with spectroscopic properties similar to that of latent rPAI-1, suggesting that denatured rPAI-1 more close ly resembles the latent rPAI-1 conformation after refolding. The spont aneous spectroscopic changes observed in rPAI-1 may reflect conformati onal transitions that are critical to the regulation of endogenous PAI -1 activity.