抄録
Alterations of polypeptide backbone structure and plasminogen activator activity of human urinary urokinase (UK) denatured with 0-6 M guanidine hydrochloride (Gdn-HCl) were investigated. The fractions of helix, β-from, β-turn and unordered structure were determined by the best-curve-fitting method based on the circular dichroic (CD) spectra at 200-240 nm to be 0.14, 0.23, 0.26 and 0.37 for M.W. 55000 form (H-UK) and 0.12, 0.17, 0.32 and 0.40 for M.W. 36000 form (L-UK), respectively. In view of the variation with Gdn-HCl concentration of ellipticity at the negative extrema (204 nm for H-UK ; 202 nm for L-UK), the denaturation process was concluded not to be a two-state transition : at 2.0-3.0M Gdn-HCl, the helical and β-form fractions were immobilized within a narrow range of 0.00-0.01 for both UK forms, corresponding to almost unchanged minimal ellipticities even with the increase of Gdn-HCl concentration. This indicates that an intermediate state (I) exists besides the native (N) and denatured (D) states. At 0.75-1.25 M Gdn-HCl, an activated state (A) intervened between N and I for H-UK but not for L-UK. The N→A transition of H-UK was an exceptional transition in that the helicity steadily increased with the enhanced minimal ellipticity. This corresponded to the observation that the activity of H-UK was potentiated by 18% during this transition. At the same Gdn-HCl concentration, L-UK reduced both its activity by 27-38% and its minimal ellipticity in contrast to H-UK. These findings show that the denaturation processes consist of "four states-three transitions" for H-UK and "three states-two transitions" for L-UK.
