Quenching of tryptophan phosphorescence in Escherichia coli alkaline phosphatase by long-range transfer mechanisms to external agents in the rapid-diffusion limit.
Quenching of the room-temperature phosphorescence of Escherichia coli alkaline phosphatase by several freely diffusing molecules was studied, each of whose absorption spectrum overlaps the long-lived emission of this protein and which therefore can quench the excited triplet state by diffusion-enhanced Förster energy transfer. The presence of additional nonresonance transfer mechanisms was also detected, from a lack of linear dependence of quenching rate on spectral overlap. The quenching agents used were the dye molecules methyl red, methyl orange, and 2-[(4-hydroxyphenyl)azo]benzoic acid, as well as the embedded heme groups of myoglobin, metmyoglobin, and the reduced and oxidized forms of cytochrome c. Quenching was found to be greatly diminished upon reduction of each acceptor, indicating that electron transfer occurs efficiently from the excited tryptophan to the oxidized form of the acceptors. The elimination of this electron transfer in the reduced form affords the opportunity to separately measure the Förster transfer rates for the heme proteins. When the transfer rate constant thus measured for myoglobin is applied to a model where both donor and acceptor proteins are taken to be spherical with both tryptophan and the heme group placed off center (a model whose quenching rate equation is newly presented here), the depth of the phosphorescent tryptophan beneath the surface of alkaline phosphatase is found to be 16 A. This value is close to the depth of tryptophan 109 (which is known to be the phosphorescent residue in alkaline phosphatase), showing that with properly chosen probes this technique is indeed valuable for distance determinations in protein structure studies.(ABSTRACT TRUNCATED AT 250 WORDS)
Mersol
J V
JV
Institute of Gerontology, University of Michigan, Ann Arbor 48109.
Steel
D G
DG
Gafni
A
A
D000469:Alkaline Phosphatase / Q000737:chemistry*; D001391:Azo Compounds; D003574:Cytochrome c Group; D004058:Diffusion; D004579:Electron Transport; D001330:Electronic Data Processing; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D006418:Heme; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D009211:Myoglobin; D014364:Tryptophan / Q000737:chemistry*
D001391:Azo Compounds; D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009211:Myoglobin; C001413:HABA; D006418:Heme; C008492:methyl red; C100258:methyl orange; D014364:Tryptophan; D000469:Alkaline Phosphatase