Determination of the Tyrosine Content of Proteins

In 1925, I published a method for the quantitative calorimetric determination of tyr0sine.l Up to that time tyrosine had been determined either by actual isolation or by calorimetric procedures that were applied to the crude hydrolysates. The calorimetric procedures employed were not certainly characteristic for tyrosine and reactions that are allowed to proceed in the presence of 95 or more per cent of compounds that are chemically similar to tyrosine are apt to be influenced by these other compounds. The calorimetric procedure devised by K. K. Koessler and myself2 is unusually characteristic for tyrosine, for, although all phenols and many other compounds give colors with diazotized sulfanilic acid in alkaline solution, tyrosine and tyramine are peculiar in that they give very little color, in dilute solution, until these solutions are rendered strongly alkaline and then treated with hydroxylamine. This color augmentation has, thus far, been observed only with tyrosine, tyramine, and with two apparently unrelated compounds, acetone and acetoacetic ester, both enols; so it is rather characteristic for tyrosine. This reaction could not, however, be employed on a crude protein hydrolysate because any amino compound, in sufficient concentration, will give a yellow color with alkaline diazotized sulfanilic acid; and that makes a reading of the purple color, due to tyrosine, practically impossible. I sought, therefore, for a method that would separate tyrosine from the bulk of other amino acids and found that when tyrosine is boiled in acetic acid solution with mercuric acetate and the cooled