Interference by Monosaccharides with the Estimation of Tyrosine and Proteins Using the Folin-Ciocalteu Phenol Reagent
The phenol color method (1)) which determines the split products soluble in trichloroacetic acid (TCA) of a standard protein, is a convenient method for the assay of proteolytic enzymes. A widely used method for the estimation of proteins is that of Lowry et al. (2). In both systems use is made of the Folin-Ciocalteu reagent (3) which yields a blue color, principally with tyrosine, tryptophan, and cysteine (4).
In addition to the compounds listed in the original description of the method (2), more recent reports have revealed other substances which interfere with the Lowry protein estimation; these include sucrose (5)) oxidized lipids (6)) and certain buffers and other chemicals (7). In this communication the influence of some monosaccharides on the estimation of tyrosine and protein by means of the Folin-Ciocalteu reagent will be discussed. This work stems from an observation that hydrolyzed sucrose gave rise to spurious tyrosine values in the assay of proteolytic enzymes by the phenol color method.
Method. Tyrosine estimation associated with proteolytic enzyme assays as used in this laboratory is as follows: The reaction mixture consists of 1 ml tyrosine 'solution, 5 ml 0.5 N NaOH, and 2 ml 1:3 Folin-Ciocalteu reagent. The absorbance is read at 750 nm after allowing the mixture to stand for 10 min.
Results. If a solution containing equal volumes of 0.25 M sucrose and 20% (w/v) TCA is assayed over a time course at 4", 23", and 37"C, there an increase in Folin-Ciocalteu reactivity.
At the higher temperatures there is a sharp increase over the first 2 hr. The color yield at 1 hr is equivalent to 0.1 wmole tyrosine/ml at 23% and 0.16 meole tyrosine/ ml at 37ยฐC. At 4ยฐC there is a slight increase over the first 2 hr, after which no further increase occurs. Fructose constitutes the chief chromogenic compound in the system. It is therefore necessary to exercise careful