Inhibition of Human Urinary Kallikrein (Urokallikrein) by Anti-Enzyme Fab

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Abstract
Monospecific rabbit IgG and Fab antibodies against human urokallikrein inhibit the proteolytic and esterolytic functions of the enzyme in a substrate dose-dependent fashion, indicating an antibody specificity for antigenic determinants at the active site of the enzyme. The specific anti-urokallikrein Fab fragments exhibited charge heterogeneity on isoelectric focusing but were oligoclonal in comparison with the distribution of the entire Fab pool. The inhibition of the kinin-generating activity of urokallikrein by the specific Fab fragments was complete at 30 sec, the earliest time interval measured. The average inhibition constant for the specific monovalent Fab fragments is the same as for the specific IgG, namely, 2.2 × 10-8 M, a finding compatible with the observations of others employing haptenic determinants. Since the molar concentrations required for comparable inhibition by anti-urokallikrein Fab were greater than the molar concentrations of anti-urokallikrein IgG, the inhibition of enzyme function by anti-urokallikrein IgG reflects both active site neutralization and additional nonspecific effects including precipitation. Analysis of the inhibition of urokallikrein kiningenerating activity by anti-urokallikrein Fab by the method of Dixon indicated the mechanism to be competitive. The additional finding that anti-urokallikrein Fab gave a dose-related inhibition of the action of urokallikrein on incremental concentrations of the synthetic substrate, α-N-p-tosyl-l-arginine methyl ester, indicated action at the active site. Analysis of these data by Dixon plots revealed competitive inhibition at the lower concentrations of Fab with an average inhibitory constant of 2.5 × 10-7 M and mixed inhibition at higher concentrations, thereby suggesting the presence of some specificities for antigenic determinants other than those at the active site. These studies demonstrate that anti-urokallikrein IgG yields a population of anti-urokallikrein Fab directed so as to neutralize proteolytic and esterolytic functions of the enzyme by a competitive mechanism.