Time resolved spectroscopy of tryptophyl fluorescence of yeast 3-phosphoglycerate kinase.
The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime tau 1 = 0.6 ns and a maximum fluorescence wavelength lambda 1max = 332 nm. The second tryptophan residue exhibits two lifetimes tau 21 = 3.1 ns and tau 22 = 7.0 ns (lambda 2max = 338 nm). In agreement with the attribution of tau 21 and tau 22 to the same tryptophan residue, the ratio beta = C21/C22 of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally to the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay tie equilibrium constant of the internal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D010735:Phosphoglycerate Kinase; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis*
D014364:Tryptophan; D010735:Phosphoglycerate Kinase