Transfer of singlet energy within trypsin.

Transfers of singlet energy within trypsin were investigated by measuring the fluorescence absorption anisotropy of its tryptophan residues. A ratio of the anisotropy of trypsin to that for N-acetyl-L-tryptophanamide was determined between 306 and 250 nm. The ratio had an average value of 0.7, whether the trypsin anisotropy was measured at 228 of 296 K. However, trypsin dissolved in 5 M guanidine hydrochloride showed little fluorescence depolarization at 228 K (the anisotropy ratio was approximately equal to 0.9). Thus, there is an extensive conformation-dependent energy transfer between tryptophans in trypsin. The ratio of anisotropies of tyrpsin at 304--270 nm was used to estimate energy transfer from tyrosine to tryptophan. Ratios of 1.8 and 1.7 were obtained at 296 K for the native and guanidinium-unfolded enzyme, respectively. The comparable value for N-acetyl-L-tryptophanamide was 1.7. This indicates that there is little transfer from tyrosine to tryptophan in trypsin at 296 K. As confirmation, the excitation wavelength dependencies of the indole fluorescence quantum yield were the same for native and unfolded trypsin. When experiments were performed at 228 K, the 304--270-nm anisotropy ratios were 2.6 for native and 2.1 for unfolded trypsin at pH2. This indicates that the efficiency of energy transfer from tyrosine to tryptophan increases at low temperatures. A photochemical source of error in the quantitation of the efficiency of energy transfer from tyrosine to tryptophan is also described.