Contribution of Each Tryptophan to the Total Fluorescence Emitted by a Protein with Multiple Tryptophan Residues Depends on the Energy of the Excitation Radiation Quantum

The structural changes induced by the addition of sodium dodecyl sulfate (SDS) in chymotrypsin and chymotrypsinogen were studied by the stopped-flow kinetic method with tryptophan fluorescence observation of the transients. Four fluorescence excitation wavelengths were used: 222, 260, 280, and 295 nm. It was found that the recorded transients were dependent on the excitation wavelength. The difference emission spectra between the complex and the free enzyme recorded for different excitation wavelengths are different. They contain a positive limb of fluorescence enhancement around 380 nm and a limb of fluorescence quenching around 340 nm. Their relative sizes depend on the excitation length, which fully explains the kinetic observations and proves that the contribution of tryptophans distributed at different sites within the protein molecule to its total fluorescence depends on the excitation wavelength. This is an important and novel finding that goes beyond the well-known fact that tryptophans distributed at different sites in the protein molecule have different fluorescence intensities.