Protein determination with trinitrobenzene sulfonate: a method relatively independent of amino acid composition

Conditions are described for precise quantitative measurement of microgram protein samples by spectrophotometric determination of the trinitrobenzene derivatives of amino acids in hydrolysates. The mean molar absorbances of individual amino acids were measured and the effective molar absorbance for use in protein measurements of 1.9 X 10(4) A M-1 cm-1 has been determined. From measurements using the trinitrobenzene sulfonate and fluorescamine reagents, and the published data on the o-phthaldialdehyde method, the molar absorption coefficients and the relative fluorescent yields are compared for the amino acids derivatives found in protein hydrolysates. The coefficients of variation for the trinitrobenzene derivatives are less than that for either the fluorescamine or the o-phthaldialdehyde derivatives. The color yields for five soluble proteins were also compared using the Lowry, Bradford, and trinitrobenzene sulfonate reagents. The results show that the described trinitrobenzene sulfonate method is more sensitive and produces a threefold smaller variation in absorbance per milligram protein than either the Lowry or the Bradford methods.