Trypsin-treated Neurospora tryptophan synthetase: II. Antigenic properties

The antigenic properties of TSase‡ that has been digested with trypsin have been examined using antisera prepared against both native and trypsin-treated TSase (digest). Although both types of antisera inhibit all TSase reactions except the synthesis of indoleglycerolphosphate from indole and glyceraldehyde-phosphate (reaction 3R), the sera may be distinguished by the pattern of inhibition. The equilibrium constant for both the enzyme/anti-enzyme reaction and the enzyme/anti-digest reaction is about 10⁻⁹M. Antigenic assays which rely on specific inhibition of TSase suggest that all the antigenic sites of the native enzyme survive tryptic treatment, but with altered combining properties. Although these alterations are detectable with either antiserum, there is a marked dependence upon time for the heterologous , digest/anti-enzyme reaction not found for the homologous digest/anti-digest reaction. Determinations of competition between trypsin-treated and native TSase for antisera indicate that trypsin-treated TSase combines more effectively with anti-digest than with anti-enzyme. Nevertheless, a sufficient excess of digest can also displace native enzyme from an enzyme/anti-enzyme complex. Quantitative precipitin analyses for the digest/anti-enzyme system show that precipitation is not complete. In Ouchterlony agar double-diffusion analyses, trypsin-treated TSase does not form a precipitin band with either anti-TSase or anti-digest. Nevertheless, digest will block the precipitation of enzyme by anti-enzyme, provided that the digest and anti-enzyme are initially mixed. The precipitin bands for enzyme/anti-enzyme and enzyme/anti-digest are confluent. These results also are compatible with the conclusion that most of the antigenic sites of the tryptophan synthetase molecule survive tryptic treatment, but with altered combining properties. It is suggested that tryptic treatment of the native enzyme modifies its configuration, and that this modification is reflected in changes in both catalytic properties and the rate of formation of antigen/antibody aggregates.