Direct kinetic evidence for triplet state energy transfer from Escherichia coli alkaline phosphatase tryptophan 109 to bound terbium.
The addition of excess Tb3+ to metal-depleted Escherichia coli alkaline phosphatase results in enhanced luminescence from enzyme-bound terbium, which increases with sample deoxygenation and exhibits a tryptophan-like excitation spectrum. Following pulsed excitation at 280 nm, the time-resolved terbium emission shows a negative prefactor associated with a submillisecond rise time, which is independent of the concentration of dissolved oxygen. The absence of a build-up phase and similarity in lifetime in the decay kinetics of directly excited (488 nm) terbium allows for the assignment of the submillisecond component in the 280 nm excited sample to bound terbium. The results of the steady state and time-resolved experiments suggest that the time evolution of alkaline phosphatase-bound terbium emission is determined by energy transfer (kET approximately 360 and 120 s-1) from the triplet state of tryptophan to terbium followed by terbium decay. This model is based on the observations that 1) the tryptophan phosphorescence lifetime (previously assigned to Trp109) corresponds to the longer component of the terbium emission and 2) the long-lived emission is enhanced, as is the Trp109 phosphorescence, by deoxygenation. An energy transfer mechanism involving the Trp109 triplet state is shown to be inconsistent with a dipole-dipole process and is best understood as a through-space electron exchange over a donor-acceptor distance of 9-10 A.
Schlyer
B D
BD
Institute of Gerontology, University of Michigan, Ann Arbor 48109, USA.
Steel
D G
DG
Gafni
A
A
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008163:Luminescent Measurements; D008433:Mathematics; D008962:Models, Theoretical; D013725:Terbium / Q000378:metabolism* / Q000494:pharmacology*; D014364:Tryptophan
D013725:Terbium; D014364:Tryptophan; D000469:Alkaline Phosphatase