Excited states of tryptophan in cod parvalbumin. Identification of a short-lived emitting triplet state at room temperature.
The fluorescence and phosphorescence spectra of model indole compounds and of cod parvalbumin III, a protein containing a single tryptophan and no tyrosine, were examined in the time scale ranging from subnanoseconds to milliseconds at 25 degrees C in aqueous buffer. For both Ca- bound and Ca-free parvalbumin and for model indole compounds that contained a proton donor, a phosphorescent species emitting at 450 nm with a lifetime of approximately 20-40 ns could be identified. A longer-lived phosphorescence is also apparent; it has approximately the same absorption and emission spectrum as the short-lived triplet molecule. For Ca parvalbumin, the decay of the long-lived triplet tryptophan is roughly exponential with a lifetime of 4.7 ms at 25 degrees C whereas for N-acetyltryptophanamide in aqueous buffer the decay lifetime was 30 microseconds. In contrast, the lifetime of the long-lived tryptophan species is much shorter in the Ca-free protein compared with Ca parvalbumin, and the decay shows complex nonexponential kinetics over the entire time range from 100 ns to 1 ms. It is concluded that the photochemistry of tryptophan must take into account the existence of two excited triplet species and that there are quenching moieties within the protein matrix that decrease the phosphorescence yield in a dynamic manner for the Ca-depleted parvalbumin. In contrast, for Ca parvalbumin, the tryptophan site is rigid on the time scale of milliseconds.
Sudhakar
K
K
Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, Philadelphia, Pennsylvania.
Phillips
C M
CM
Williams
S A
SA
Vanderkooi
J M
JM
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000737:chemistry; D005399:Fishes; D049449:Luminescence; D010320:Parvalbumins / Q000737:chemistry* / Q000528:radiation effects; D010777:Photochemistry; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry / Q000528:radiation effects
D010320:Parvalbumins; D014364:Tryptophan; D002118:Calcium