Picosecond-resolved fluorescence spectra of D-amino-acid oxidase. A new fluorescent species of the coenzyme.
Protein dynamics of D-amino-acid oxidase in the picosecond region was investigated by measuring time-resolved fluorescence of the bound coenzyme, FAD. The observed nonexponential fluorescence decay curves were analyzed with four-exponential decay functions. The fluorescence lifetimes at the best fit were 26.6 +/- 0.7 ps, 44.0 +/- 4.2 ps, 177 +/- 11 ps, and 2.28 +/- 0.21 ns at 20 degrees C and 25.2 +/- 3.0 ps, 50.3 +/- 8.7 ps, 228 +/- 27 ps, and 2.75 +/- 0.33 ns at 5 degrees C. Component fractions with the shortest lifetime, ca. 26 ps, were always negative and close to -1. The other fluorescent components of the lifetimes, ca. 47 ps, 200 ps, and 2.6 ns, with positive fractions were assigned to different forms of the enzyme including the dimer, the monomer, and free FAD dissociated from the enzyme. Measurements of the time-resolved fluorescence spectra revealed that the maximum wavelengths of the spectra shifted toward shorter wavelength by 65 nm at 20 degrees C and 36 nm at 5 degrees C within 100 ps after pulsed excitation. The remarkable blue shift was not observed in free FAD. The first spectra immediately after the excitation of the enzyme exhibited maximum wavelengths of 584 nm at 20 degrees C and 557 nm at 5 degrees C. The fluorescence spectra obtained at times later than 100 ps are in good agreement with the one obtained under steady-state excitation of D-amino-acid oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)
Tanaka
F
F
Mie Nursing College, Tsu, Japan.
Tamai
N
N
Yamazaki
I
I
D000818:Animals; D003067:Coenzymes; D003605:D-Amino-Acid Oxidase; D005182:Flavin-Adenine Dinucleotide; D013050:Spectrometry, Fluorescence
D003067:Coenzymes; D005182:Flavin-Adenine Dinucleotide; D003605:D-Amino-Acid Oxidase