Long-range electron exchange measured in proteins by quenching of tryptophan phosphorescence.

Abstract

Ten In earlier work, we showed that the phenomenon of roomtemperature protein phosphorescence, though previously seen only rarely (1, 2), in fact can be found in the great majority of proteins (3). Of 40 proteins surveyed, 29 were found to exhibit phosphorescence in aqueous solution at room temperature with a wide range of lifetimes-between about 0.5 msec and 2 sec. The central requirement for the observation of protein phosphorescence in solution is to reduce dissolved oxygen to a sufficiently low level, since oxygen can efficiently quench the excited tryptophan triplet state, even when the tryptophan is buried in the protein matrix (3-6).A subsequent study (7) revealed that a variety of smallmolecule agents in addition to dioxygen can quench the phosphorescence of protein tryptophans. Surprisingly, even though the phosphorescent tryptophans are well buried within the protein, the quenching efficiency of most of the agents tested (those larger than three atoms in size) was found to be independent of the size and polarity of the quenching agent. This indicates that the quenching process does not involve the penetration of these quenchers through the protein matrix to the position of the buried tryptophan. Although the sensitivity of the different proteins to quenching was found to be spread over a wide range, the various agents tested all quenched any given protein with similar efficiency, indicating that the quenching reaction is determined by some property of the individual tryptophan or the protein itself rather than by the particular quenching agent used. Finally, the quenching rate was essentially independent of solution viscosity. This rules out the possibility of a protein-opening reaction that might transiently bring the buried tryptophan into contact with solvent and the added quenchers. All these properties might be explained, it was noted, if the quenching process involves long-range electron transfer occurring on a long time scale, so that the quenchers in solution are effectively in the rapid diffusion limit.The present work represents an attempt to identify the protein parameters that determine the sensitivity of its tryptophans to the quenching process. We compared various structural parameters of 10 different proteins with their ability to be quenched by four different small-molecule agents. The results show that quenching rate constants decrease exponentially with the distance of the tryptophan from the protein surface, consistent with an electronexchange reaction in the rapid diffusion limit. Analysis of the data then provides an estimate of the dependence of electrontransfer rate on distance when the intervening space is filled with averaged protein matrix. MATERIALS AND METHODSGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) from porcine muscle, Pronase type XIV from Streptomyces griseus, and protease type X from Bacillus thermoproteolyticus rokko were obtained from Sigma. Nuclease from Staphylococcus aureus was a gift of E. E. Lattman (Baltimore). The sources of other proteins and su...

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins¶

“…For example, free Cys in the solvent could not quench the phosphorescence of W59 of RNaseT1, which is merely 0.2nm from the aqueous interface (3). The bimolecular rate constant derived from the above expression (30) is 5 × 10 2 M −1 • s −1 , a value comparable to the detection limit for that system (3). Likewise, the lifetime of W48 in azurin (1/τ 0 ∼ 1 s −1 ) is not affected by the removal of the disulfide bridge C23‐C26 placed at a distance of ∼ 1.1 nm [k(1.1nm) = 0.003 s −1 ] (P. Cioni, unpublished work).…”

Light-induced aggregation of type I soluble tumor necrosis factor receptor

“…Following light absorption at 302 nm, whether the triplet excited state of tryptophan ( 3 Trp)41–46 was potentially involved in the observed photo‐reactions of sTNF‐R1 was investigated. This was done by evaluating the effects of additives, acrylamide and Na‐azide, quenchers of the triplet excited states of tryptophan 45, 47.…”

Conformational changes in proteins induced by dynamic associations

“…To verify how specific to LADH this phenomenon is, we studied the behaviour of other proteins selecting GraPDH and LDH for their amenable phosphorescence emissions and for being Vanderkooi et al (1990). AP = alkaline phosphatase.…”