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

Abstract

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.

Fluorescence detected circular dichroism

“…with 4c or d, nor were products of reaction with 2 mol of oxygen (3) observed (by nmr) under these conditions. Dioxetanes 5a-d chemiluminesce when CHC13 solutions are warmed with 9,10-diphenylanthracene or 9,10dibromoanthracene.…”

Circularly polarized luminescence spectroscopy

“…This may be examined in a bit more detail by performing a simple vibronic analysis of R®(g-*n) and R®(cp-n) within the Born-Oppenheimer (B-O) approximation. Ignoring all "hot-band" vibronic contributions in both absorption and emission (7 = 0 K), we may write: (82) and Re(g*~ri) = £fie(gr'-nO…”

Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases

“…This technique yields information about the conformations of chiral systems in their electronically excited states in the same way that CD reports on the ground state conformation (Steinberg, 1975(Steinberg, , 1978. Since CPL combines the sensitivity of both optical activity and fluorescence techniques, it was found to be extremely sensitive to subtle conformational details of chiral systems (Schlessinger & Steinberg, 1972;Gafni et al, 1973;Gafni & Steinberg, f From the Chemical Physics Department, Weizmann Institute of Science, Rehovot, Israel. Received October 18, 1977. 1 Abbreviations used: NADH, reduced /3-nicotinamide adenine dinucleotide; NMNH, reduced nicotinamide mononucleotide; eNAD+, nicotinamide 1 ,!V6-ethenoadenine dinucleotide; LDH, beef heart lactate dehydrogenase; LADH, horse liver alcohol dehydrogenase; GPDH, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase; CD, circular dichroism; CPL, circular polarization of luminescence; IBA, isobutyramide.…”