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. 1 I3 114 M. J. KRONMAN and L. G . HOLMES EXPERIMENTAL Materials.With the exceptions noted below, enzymes and other proteins used in this study were the purest grade available from Worthington Biochemical and Sigma and were not subjected to further purification. a-Lactalbumin was prepared by the method of Robbins and Kronman[3] and the two genetic forms of P-lactoglobulin[4,5] A and B, were obtained through the courtesy of Dr. R. Townend. Crystallized bovine and human serum albumin were freed of the fatty acid impurity by charcoal treatment according to the method of Chen [6]. N-Acetyl tryptophan ethyl ester and N-acetyl tyrosine ethyl esters were products of Mann Research Laboratories, Inc. Urea, a Baker product, was crystallized from alcohol. Eastman Guanidine-HC1 was recrystallized according to Nozaki and Tanford[7]. In later stages of this work 'ultrapure' guanidine-HC1 and urea as obtained from Mann Research Laboratories, Inc. were employed. Both of these materials as well as the guanidine-HC1 and urea recrystallized in our laboratory had very low absorption in the U.V. region. All other chemicals of reagent grade were used without further purification. Glass distilled water was used in preparing all solutions. Dithiothreitol (Cleland's reagent) was obtained from Calbiochem and ethylenimine was purchased from Doe and Ingalls.Preparation of denatured and reduced denatured proteins. Proteins were denatured by dilution of a suitable aliquot in buffer with a concentrated solution of guanidine-HC1 such that the final concentration was 6 M . Fluorescence and ancillary absorption measurements were made with appropriate dilutions of 6 M guanidine-HC1 no less than 3 hr after initiation of denaturation.Reduction of the disulfide bridges was effected ...

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