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 adsorption spectrum overlaps the long-lived emission of this protein and which therefore can quench the excited triplet state by diffusion-enhanced Forster 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 trytophan to the oxidized form of the acceptors. The elimination of this electron transfer in the reduced form affords the opportunity to separately measure the Forster 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 trytophan 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 angstrom. This value is close to the depth of trytophan 109 (which is known to be the phosphoresecent residue in alkaline phosphatase), showing that with properly chosen probes this technique is indeed valuable for distance determinations in protein structure studies. The distance calculated from cytochrome c data was found to vary among different buffers and also to depend on buffer concentration, changing from 8 to 12 angstrom upon increase of Tris.HCl concentration from 50 mM to 1 M. This reflects the need for a model for cytochrome c which better represents its shape and electrostatic properties.