ANALOGS OF HUMAN PLASMINOGEN THAT ARE LABELED WITH FLUORESCENCE PROBES AT THE CATALYTIC SITE OF THE ZYMOGEN - PREPARATION, CHARACTERIZATION, AND INTERACTION WITH STREPTOKINASE

Fluorescent analogs of the proteinase zymogen, plas minogen (Pg), whic h are specifically inactivated and labeled at the catalytic site have been prepared and characterized as probes of the mechanisms of Pg acti vation. The active site induced non-proteolytically in Pg by streptoki nase (SK) was inactivated stoichiometrically with the thioester peptid e chloromethyl ketone, -alpha-[(acetylthio)acetyl]-(D-Phe)-Phe-Arg-CH2 Cl; the thiol group generated subsequently on the incorporated inhibit or with NH2OH was quantitatively labeled with the fluorescence probe, 2-((4'-iodoacetamido) anilino)naphthalene-6-sulfonic acid; and the lab eled Pg was separated from SK. Cleavage of labeled [Glu]Pg(1) by uroki nase-type plasminogen activator (uPA) was accompanied by a fluorescenc e enhancement (Delta F-max/F-o) of 2.0, and formation of 1% plasmin (P m) activity. Comparison of labeled and native [Glu]Pg(1) as uPA substr ates showed that activation of labeled [Glu]Pg(1) generated [Glu]Pm-1 as the major product, while native [Glu]Pg(1) was activated at a faste r rate and produced [Lys]Pm-1 because of concurrent proteolysis by pla smin, When a mixture of labeled and native Pg was activated, to includ e plasmin-feedback reactions, the zymogens were activated at equivalen t rates. The lack of potential proteolytic activity of the Pg derivati ves allowed their interactions with SK to be studied under equilibrium binding conditions. SK bound to labeled [Glu]Pg(1) and [Lys]Pg(1) wit h dissociation constants of 590 +/- 110 and 11 +/- 7 nM, and fluoresce nce enhancements of 3.1 +/- 0.1 and 1.6 +/- 0.1, respectively. Charact erization of the interaction of SK with native [Glu]Pg(1) by the use o f labeled [Glu]Pg(1) as a probe indicated a similar to 6-fold higher a ffinity of SK for the native Pg zymogen compared to the labeled Pg ana log. Saturating levels of epsilon-aminocaproic acid reduced the affini ty of SH for labeled [Glu]Pg(1) by similar to 2-fold and lowered the f lu orescence enhancement to 1.8 +/- 0.1, whereas the affinity of SK fo r labeled [Lys]Pg(1) was reduced by similar to 98-fold with little eff ect on the enhancement. These results demonstrate that occupation of l ysine binding sites modulates the affinity of SK for Pg and the change s in the environment of the catalytic site associated with SK-induced conformational activation. Together, these studies show that the label ed Pg derivatives behave as analogs of native Pg which report function ally significant changes in the environment of the catalytic site of t he zymogen.