It has long been reported that a purple or red color can be produced when tyrosine is reacted with l-nitroso-2-naphtho'l
(1). The use of this reaction for the calorimetric determination of tyrosine was then developed (2, 3). The timing required for the crucial heating step and the instability of the red compound formed in the earlier procedures led to the currently used modification by Udenfriend and Cooper (4). Following their method, a stable yellow product is formed after prolonged heating of tyrosine or tyramine with excess nitrosonaphthol at 55" in dilute nitric acid. The more water-soluble, colored product can be separated from unreacted nitrosonaphthol, which is removed by extraction with 1,2-dichloroethane. However, the similarity between the spectral characteristics in the visible and near-ultraviolet range of the tyrosine-nitrosonaphthol product and of flavin makes difficult the quantitative assessment of one in the presence of the other.
In this report, we describe a simple modification of the tyrosine assay which leads to the accurate determination of this amino acid in the presence or absence of flavin. This is especially useful for quantitating tyrosine in hydrolyzates from flavoproteins. Materials and Methods. n-Tyrosine was obtained from Nutritional Biochemicals
Corp. 1-NitrosoS-naphthol and 1,2-dichloroethane were from Eastman Organic Chemicals. A commercial grade of the monosodium salt of flavin mononucleotide (FMN) was from Sigma Chemical co.
Essentially, the method described by Udenfriend and Cooper (4) was used with and without modification. In some experiments, this consisted only in first dissolving the tyrosine (0.276 qole) in 2 ml 3 N HCl instead of water as normally done. After addition of 1 ml each of 0.1% 1-nitroso-&naphthol in 95% ethanol and 1:s nitric acid containing 0.5