Singlet Energy-Transfer Studies on Associating Protein Systems. Distance Measurements on Trypsin, α-Chymotrypsin, and Their Protein Inhibitors

Singlet-singlet energy transfer has been measured between fluorescent dyes covalently attached to single proteins and to trypsin-trypsin inhibitor complexes. To maximize the probability of surface labeling, Celite-bound reactive dyes were used. These included fluorescein and Rhodamine B isothiocyanates and dansyl chloride. The Celite technique permits the production of heavily labeled proteins which retain nearly full activity. The experimental energy-transfer results are in excellent agreement with calculations which assume random surface labeling of spherical proteins with known anhydrous radii and a Poisson distribution of the degree of labeling. The experiments on complexes of trypsin with lima and soya bean trypsin inhibitors show that singlet energy transfer is a practical and simple method for determining the distance between specific proteins in a large protein complex. Extrinsic fluorescence labels are capable of providing semiquantitative distance information. Preliminary energy transfer results indicate that trypsin and chymotrypsin form a stable complex at low concentration. The experimental protocol and interpretive framework should be easily generalizable to a variety of complex systems. This rapid and sensitive technique will be of general utility in studies of the size, spatial arrangement, stoichiometry, and kinetics of associating macromolecules.