Time-resolved circularly polarized protein phosphorescence.
The existence of circular polarization in room-temperature protein phosphorescence is demonstrated, and time-resolved circularly polarized phosphorescence (TR-CPP) is used to characterize unique tryptophan environments in multitryptophan proteins. Circularly polarized luminescence studies provide information regarding the excited state chirality of a lumiphore which can be used to extract sensitive structural information. It is shown by time resolving the circular polarization that it is possible to correlate the excited state chirality with unique decay components in a multiexponential phosphorescence decay profile. The present study presents a concurrent analysis of room-temperature time-resolved phosphorescence and TR-CPP of bacterial glucose-6-phosphate dehydrogenase as well as those of horse liver alcohol dehydrogenase. Only one of the two tryptophan residues per subunit of dimeric alcohol dehydrogenase is believed to phosphorescence, while the dimeric glucose-6-phosphate dehydrogenase has eight tryptophan residues per subunit and shows a corresponding complexity in its phosphorescence decay profile. The anisotropy factor [g(em) = delta I/(Itotal/2); delta I = Ileft circular-Iright circular] for alcohol dehydrogenase is time independent, suggesting a unique excited state chirality. The phosphorescence decay of glucose-6-phosphate dehydrogenase can be well fitted with four exponential terms of 4, 23, 76, and 142 msec, and the TR-CPP of this enzyme shows a strong time dependence that can be resolved into four individual time-independent anisotropy factors of -4.0, -2.1, +6.5, and +6.9 (x10(-3)), each respectively associated with one of the four lifetime components. These results demonstrate how the use of TR-CPP can facilitate the study of proteins with multiple lumiphores.
Schauerte
J A
JA
Institute of Gerontology, University of Michigan, Ann Arbor 48109.
Steel
D G
DG
Gafni
A
A
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000737:chemistry; D000818:Animals; D004798:Enzymes / Q000737:chemistry*; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry; D006736:Horses; D007700:Kinetics; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D008433:Mathematics; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013997:Time Factors; D014364:Tryptophan
D004798:Enzymes; D046911:Macromolecular Substances; D011506:Proteins; D014364:Tryptophan; D000426:Alcohol Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D000469:Alkaline Phosphatase