Quenching of room temperature protein phosphorescence by added small molecules (original) (raw)

A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) > parvalbumin = lactoglobulin = ribonuclease TI > liver alcohol dehydrogenase > aldolase > Pronase = edestin > azurin > alkaline phosphatase. Quenching rate constants for O2 and C O are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to lo5 lower for H2S and CS2. Larger and more polar quenchers-including organic thiols, conjugated ketones and amides, and anionic species-were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer. In addition, the strong apparent correlation between degree of burial and protection against quenching is consistent with the sharp distance dependence expected for a long-range transfer mechanism. Since the conditions for resonance energy (Forster)