Circular Polarization of Fluorescence of Probes Bound to Chymotrypsin. Change in Asymmetric Environment upon Electronic Excitation

The circular polarization of fluorescence is related to the conformational asymmetry of the emitting molecule in the first singlet excited state in the same way that circular dichroism is related to the conformational asymmetry of the absorbing molecule in the electronic ground state. By measurement of these optical phenomena, the induced asymmetry of two chromophores bound to chymotrypsin (EC 3.4.4.5) when in the ground state was compared with the induced asymmetry of the ligands when in the excited state. The two chromophores studied were 2-p -toluidinylnaphthalene-6-sulfonate (TNS), bound at a specific site which is not the active site of the protein, and an anthraniloyl group, bound at the active site of the enzyme. Both chromophores showed a change in induced asymmetry upon electronic excitation, the effect being particularly large in the case of the TNS chromophore. It is thus concluded that the orientation of TNS in the binding site, its freedom of rotation in its site, the strength of binding, or even the site of binding of the dye to the protein might have changed upon electronic excitation.