The Fluorescence of Native, Denatured and Reduced‐denatured Proteins*

Abstract— (1) By excitation at 295 nm tryptophan fluorescence from 17 proteins was examined free of contributions from tyrosine. The tryptophan quantum yields for native proteins were both higher and lower than that of the free amino acid and spanned a 5‐fold range. No simple relationship was apparent between ‘exposure’ of tryptophyl side chains in proteins and the magnitude of the quantum yield or of the position of the emission maximum. (2) Denaturation of these proteins in 6 M guanidine hydrochloride considerably narrowed the range of values of yields ( ca 0.1 to 0.17). While reduction of the disulfide bridges altered the yield of several proteins, it appeared to have no general effect on narrowing the range observed with a group of proteins, suggesting that either: (a) the amino acid sequence around a tryptophan in a disrupted peptide chain influences the yield, or (b) ‘residual’ three dimensional structure persists in reduced denatured proteins. (3) It was possible to demonstrate using both 280 and 295 nm excited spectra that tyrosine fluorescence, while weak, is generally present in proteins. These data also showed that transfer of the excited state from tyrosine to tryptophan is a common occurrence in native proteins and occurs with very high efficiency in a number of cases.