Effects of bound monoclonal antibodies on the decay of the phototransformation intermediates I700(1,2) from native Avena phytochrome.

The kinetics of the microsecond phototransformation intermediates of 124 kDa Avena phytochrome (I700(1,2) were studied in the presence of bound monoclonal antibodies at various temperatures. A global analysis was applied to the decays at all wavelengths at each temperature in order to derive the rate constants and the decay-associated spectra of the three decay components. Monoclonal antibodies bound to specific epitopes altered the Arrhenius parameters of both I700(1,2) decay components. The strongest influence on these parameters was observed with OAT 8 (epitope between residues 624 and 686), which decreased by more than 50% the activation parameters of both components. This decrease is interpreted to result from an increased flexibility induced by this antibody in the ground state or in the transition state of bonds changing during the decay of both I700 transients. Thus, the OAT 8 epitope appears to be functionally important during the decay of the I700(1,2) intermediates. For the case of I700(1), bound OAT 23 and OAT 25 (epitopes between residues 1 and 66) reduced even further the relatively small flexibility of these bonds in the red light-absorbing form of phytochrome (Pr) without antibodies, as reflected by the high preexponential factors for its decay. This resulted also in higher activation energies for this decay in the presence of the antibodies. Thus, the amino-terminus should act as a rigid spacer of the chromophore cavity without affecting it during the microsecond transformation, because the Arrhenius parameters for these decays are similar to those for small phytochrome. The possible implications of the influence of the various antibodies on the bleaching remaining after the decay of I700(1,2) are discussed.