Second derivative fluorescence spectra of indole compounds.
The fluorescence emission spectrum of N-acetyl tryptophan amide (NATA) in 20 mM K-phosphate buffer, pH 7.5, with excitation at 295 nm, when subjected to second derivatization, showed two troughs at 340 1.0 nm (A) and 358.5 1.0 nm (B). Linear dependence of derivative intensities at A and B was observed with increasing NATA concentration between 0-30 nM but the intensity ratio (B/A), termed R, was found to be invariant at 0.70 0.05. R remained unaffected with variation of the pH (4-10), temperature (15-70 degrees C), salt concentration (0-2 M NaCl), and excitation wavelength between 280-300 nm. A 50-fold molar excess of N-acetyl tyrosine over 10 nM NATA and inclusion of a quencher like 0.8 M acrylamide, 0.4 M potassium iodide or trichloroethanol had no effect on R. It was, however, linearly dependent on the polarity of the solvent-in 1,4-dioxane it became 0.07 0.05. Derivative spectra of tryptophans of proteins largely resembled that of NATA. Low R values of between 0.02-0.34 were observed for proteins under native conditions, which is consistent with the general buried character of tryptophan residues. R increased to 0.6-0.9 after unfolding with denaturants or extensive proteolysis and decreased to close to the original value after refolding. The equilibrium unfolding transitions of proteins expressed as R largely resembled the transitions measured using other physical parameters. R appears to be a more sensitive index for monitoring the hydrophobic environment of tryptophans in protein compared to parameters like emission maxima or intensity of underivatised spectra.
Nayar
Suprabha
S
Division of Protein Engineering, Indian Institute of Chemical Biology, Raja S.C. Mallick Road, Jadavpur, Calcutta-700032, India.
Brahma
Amrita
A
Mukherjee
Chaitali
C
Bhattacharyya
Debasish
D
D020106:Acrylamide / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
D011506:Proteins; D020106:Acrylamide; C016952:N-acetyltryptophanamide; D014443:Tyrosine; D014364:Tryptophan