Second derivative fluorescence spectroscopy of tryptophan in proteins.
The second derivatives of N-acetyl-L-tryptophan amide (AcTrpNH(2)) fluorescence spectra were characterised in order to describe changes in the tryptophan environments of proteins. This tryptophan model compound was studied in several media with different degrees of hydrophobicity. The effect of tyrosines on the derivative spectra was also determined in situations in which both tyrosine and tryptophan were excited. An analysis of fluorescence second derivative spectra suggests that AcTrpNH(2) fluorescence emission is composed of two main bands. Increasing solvent polarity resulted in a red-shift by both bands and a relative increase in the emission efficiency of the shortest wavelength band. The applicability of fluorescence second derivative is shown through several examples. Turbidity observed in whole membrane extracts, for example, is eliminated by using second derivative spectra. Melittin, human and bovine serum albumins and the carboxypeptidase-PCI complex were studied as examples of the use of fluorescence second derivative spectroscopy to monitor changes in structural characteristics when these proteins were subjected to various transitions.
Mozo-Villarías
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Unitat de Biofísica, Departament de Ciencies Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, Av. Rovira Roure 44, E-25198 Lleida, Spain. angel.mozo@cmb.udl.es
D000818:Animals; D002417:Cattle; D006801:Humans; D008555:Melitten / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012997:Solvents; D013050:Spectrometry, Fluorescence / Q000379:methods* / Q000706:statistics & numerical data; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
D011506:Proteins; D012709:Serum Albumin; D012997:Solvents; D008555:Melitten; C016952:N-acetyltryptophanamide; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D014364:Tryptophan