Time-resolved phosphorescence of proteins: structural studies using energy transfer in the rapid diffusion limit

Ari Gafni; Joseph V. Mersol; Duncan G. Steel

doi:10.1117/12.17721

1 May 1990 Time-resolved phosphorescence of proteins: structural studies using energy transfer in the rapid diffusion limit

Ari Gafni, Joseph V. Mersol, Duncan G. Steel

Author Affiliations +

Proceedings Volume 1204, Time-Resolved Laser Spectroscopy in Biochemistry II; (1990) https://doi.org/10.1117/12.17721
Event: OE/LASE '90, 1990, Los Angeles, CA, United States

Abstract

Room temperature phosphorescence emitted by tryptophan residues in deoxygenated aqueous solutions of proteins is extremely sensitive to the environment of these residues and can be utilized for the detailed study of protein structure and dynamics. The long decay time of their triplet state makes the phosphorescent tryptophans suitable donors for resonance energy transfer in the rapid diffusion limit. As shown by Stryer et al. (Ann. Rev. Biophys. Bioeng. 11, 203 (1982)) proper data analysis can then yield the distance of closest approach between the donor-acceptor pair. This method can thus allow one to map the distances of phosphorescent tryptophans from the surface of the protein. In the present study a laser-based photon counting system was used to follow the room-temperature phosphorescence decays of alkaline phosphatase and horse liver alcohol dehydrogenase and to study the quenching of their triplet states by several molecules whose absorption spectra overlap the long-lived emission of these proteins. The results demonstrate the potential applicability of these measurements for the mapping of phosphorescent tryptophan residues and confirm the phosphorescence of alkaline phosphatase to originate in Trp-109. Limitations to the applicability of the energy-transfer approach arise from quenching mechanisms which compete with resonance transfer. Two such processes - electron transfer and exchange interactions - are discussed.

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Ari Gafni, Joseph V. Mersol, and Duncan G. Steel "Time-resolved phosphorescence of proteins: structural studies using energy transfer in the rapid diffusion limit", Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); https://doi.org/10.1117/12.17721

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