Fluorometric Demonstration of Tryptophan in Dentin and Bone Protein

Tryptophan is thought to be absent, or at least difficultly demonstrated, in most calcified proteins, collagens, and gelatins. An exception appears to be human tooth enamel, which contains about 0.1 per cent of this amino acid.' Human dentin and bone protein are apparently free of tryptophan.2 Piez5 has reported the absence of tryptophan in dentin protein and in a series of other collagens6 but indicated later that no intensive effort was made to confirm these findings.t Spies and Chambers7 were able to demonstrate minute quantities of tryptophan (0.01%G) in gelatin using optimal conditions of the p-dimethylamino-benzaldehyde-indole oxidation reaction. Following these same procedures, we were unable to show the presence of tryptophan in gelatin or dentin protein. Bone protein reacted to give the barest hint of color (O.D 590m = 0.006). an optical density well within the range of instrument error alone. Notwithstanding, the ultraviolet fluorescence of bone and dentin protein and some of their diand tri-peptides at pH 11.0 have suggested the presence of structural indole nuclei or other cyclic groups with emission bands between 350 and 415 mg.8 Tryptophan and tvrosine display distinct emission maxima at 343 and 300 + 5 m/i at various pH's, respectively, when excited by U.V. light at about 275 m/i. Direct identification and quantification of these two amino acids by measuring fluorescence of an intact protein molecule is difficult since tyrosine is known to transfer its absorbed energy to tryptophan even when the latter is in disproportionately small quantities.9 Consequently, complete basic hydrolysis (tryptophan is intrinsically degraded in strong acid) must be performed prior to fluorescent measurements. Duggan and Udenfriend' have successfully quantified tyrosine and tryptophan content of various proteins after complete basic hydrolysis, by the intensity of fluorescence taken at optimum excitation and emission wavelengths. The method detected tryptophan in concentrations as low as 0.1 -y/ml. In light of our fluorescent data obtained from calcified protein solutions,8 it appeared attractive to reinvestigate the possible presence of tryptophan, by fluorometric means, in basic hydrolysates of bone and dentin protein.