DETECTION OF A CATALYTIC ANTIBODY SPECIES ACYLATED AT THE ACTIVE-SITEBY ELECTROSPRAY MASS-SPECTROMETRY

The chemical interactions between a catalytic antibody Fv fragment and ester substrates were examined using pneumatically assisted electrosp ray (ion spray) mass spectrometry. Upon addition of the p-nitrophenyl ester substrate to the antibody fragment, an antibody fragment species that represents approximately 8% of the total Fv concentration is cle arly observed in the electrospray spectrum. The observed increase in m olecular weight of the Fv fragment corresponds to the mass of the acyl group of the substrate. Formation of the acyl-Fv species is blocked b y preincubation of the antibody fragment with hapten inhibitor, sugges ting that the acyl linkage involves a residue in the active site of th e antibody. The acyl-Fv species is not observed when the corresponding p-chlorophenyl ester substrate is used, indicating that the level of this species is dependent on the leaving group of the substrate, The a cylated species is not observed for a site-directed mutant lacking cat alytic activity, His L91 Gin, The present results are consistent with modeling studies of the structure of the Fv fragment and provide stron g confirmatory evidence for the multistep kinetic mechanism previously proposed for this antibody.