An improved colorimetric method for the quantitative estimation of thymine.

The Hunter (1) procedure was originally developed for the calorimetric d&&ion of minute amounts of thymine. In this procedure thymine is coupled under air with p-diazobenzenesulfonic acid in carbonate solut.ion, and a red color is subsequently produced by the addition of sodium hydroxide and hydroxylamine. Thymine was determined by this means in hydrolysates of dcsoxypentose nucleic acids (DNA) by Woodhouse (2, 3), by Pircio and Cerecedo (4), and by Day et al. (5). The original method, however, was found to permit only semiquantitative measurements of thymine (6). The reasons for the analytical limitations of the method were demonstrated by a study (6) of the Hunter reaction. First, it was shown that under air at 20” the coupling of thymine wit,h the diazo reagent required several hours for completion, but that under oxygen at 30” coupling was complete in 20 minutes. In either case, however, a certain amount of coupling of the diazo reagent with bot.h cytosine and uracil occurred during the time required for complete coupling wit,h thymine. Second, it was shown that the red color formed by the addition of alkali and hydroxylamine was transient and that t.rue maximum values of optical density could therefore not be determined. The hydroxylamine was shown to be largely responsible for color instability. In the improved procedure, consequently, the coupling reaction for thymine is carried out under pure oxygen. The diazo reagent of Koesslcr and Hanke (7) is employed at twice the concentration used in previous procedures (l-5) to insure the complete coupling of thymine, even in the presence of cytosine and uracil. A mixt.ure of glycerol and sodium hydroxide solution is substituted for hydroxylamine to give an intense and stable red color. The values of optical density of the colors produced with cytosine and uracil are nullified by appropriate blanks when these pyrimidines appear as contaminants in t.hyminc solutions. This modified procedure provides a much more reliable method for the quantitative estimation of thymine.