Contribution of\nEach Tryptophan to the Total Fluorescence\nEmitted by a Protein with Multiple Tryptophan Residues Depends on\nthe Energy of the Excitation Radiation Quantum

The structural changes induced by the addition of sodium\ndodecyl\nsulfate (SDS) in chymotrypsin and chymotrypsinogen were studied by\nthe stopped-flow kinetic method with tryptophan fluorescence observation\nof the transients. Four fluorescence excitation wavelengths were used:\n222, 260, 280, and 295 nm. It was found that the recorded transients\nwere dependent on the excitation wavelength. The difference emission\nspectra between the complex and the free enzyme recorded for different\nexcitation wavelengths are different. They contain a positive limb\nof fluorescence enhancement around 380 nm and a limb of fluorescence\nquenching around 340 nm. Their relative sizes depend on the excitation\nlength, which fully explains the kinetic observations and proves that\nthe contribution of tryptophans distributed at different sites within\nthe protein molecule to its total fluorescence depends on the excitation\nwavelength. This is an important and novel finding that goes beyond\nthe well-known fact that tryptophans distributed at different sites\nin the protein molecule have different fluorescence intensities.