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.
ASJC Scopus subject areas