Proton transfer dynamics in the nonhomogeneous electric field of a protein.
By adsorption of pyranine (8 hydroxypyrene 1, 3, 6 trisulfonate) to lysozyme we create on the positively charged protein a fluorophoric site with a total charge of -3. Photo dissociation of the dye's hydroxyl proton changes its absorption and fluorescence spectrum, permitting a continuous monitoring of the reprotonation dynamics. Absorbance measurements in the microsecond time scale monitor how the bulk protons penetrate the Coulomb cage of the bound dye. Time-resolved fluorescence monitors how the proton is escaping out of the Coulomb cage of the bound dye. These probe reactions were studied with a series of dye-enzyme complexes where the number of free carboxylate was reduced by amidation, increasing the total charge of the complex from +5 to +12.6. The time-resolved measurements demonstrate the complexity of the electric field in the immediate vicinity of the dye. It is consistent with a negative potential wall (of the anion) surrounded by a positive potential wall of proteinaceous moieties.
Yam
R
R
Laser Laboratory for Fast Reactions in Biology, Department of Biochemistry, Tel Aviv University, Israel.
Nachliel
E
E
Kiryati
S
S
Gutman
M
M
Huppert
D
D
D000327:Adsorption; D001190:Arylsulfonates / Q000737:chemistry*; D001665:Binding Sites; D004563:Electrochemistry; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation; D011522:Protons; D013997:Time Factors
D001190:Arylsulfonates; D011522:Protons; D009113:Muramidase; C005047:pyranine