The effect of interaction between human urokinase and its competitive inhibitor, N alpha-benzoyl-L-arginine amide, on reduction of a specific SS bond related to enzymatic activity.

The 55- (H-UK) and 36-kDa forms (L-UK) of human urinary urokinase lost most of esterase activity toward acetyl-glycyl-L-lysine methyl ester upon reductive cleavage of 3 SS bonds with dithiothreitol in the presence of the competitive inhibitor, N alpha-benzoyl-L-arginine amide (BAA), bound to polyacrylyl azide with C16N3-arm (PAA) at 0.3 M guanidine, a threshold point of the native state where a protein-denaturating transition began. One of the 3 SS bonds was protected from reduction, with an unaltered activity, under the similar conditions except for replacement of BAA-PAA conjugate by glycine-PAA conjugate. This "specific" SS bond was reduced and, after the other SH groups produced were blocked with iodoacetamide (IAM), selectively reoxidized, which resulted in complete reactivation. The intact B-chain isolated from H-UK was completely inactivated when its specific SS bond was reduced and selectively alkylated with IAM after the other SH groups were reversibly blocked with 5, 5'-dithiobis (2-nitrobenzoic acid), which was finally removed. The results indicate that a single specific SS bond is essential for retaining a conformation necessary to activity exhibition.