No abstract

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) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO 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 10(5) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Experiments were carried out to measure the effect of concentrations of glycerol on H-exchange (HX) rates by using myoglobin as a test protein. Concentrated glycerol has only a small slowing effect on the HX kinetics of freely exposed amides, studied in a small molecule model (acetamide). Larger effects occur in structured proteins. The effect of solvent glycerol on different parts of the HX curve of myoglobin was studied by use of a selective "kinetic labeling" approach. Concentrated glycerol exerts an apparently reverse effect on protein H exchange; the faster exchanging "surface" protons are least affected, while the slower and slower amide NH is further slowed by larger and larger factors. These results seem inconsistent with solvent penetration models which generally visualize slower and slower protons as being placed, and undergoing exchange, farther and farther from the solvent-protein interface. On the other hand, the results are as expected for the local unfolding model for protein H exchange since concentrated glycerol is known to stabilize proteins against unfolding. In the local unfolding model, slower exchanging protons are released by way of higher energy and therefore generally larger, unfolding reactions. Larger unfoldings must be more inhibited by the glycerol effect.