The quantification of protein amino groups by the trinitrobenzenesulfonic acid method: a reexamination

Trinitrobenzenesulfonic acid (TNBSA) is the reagent in a well-known method for quantification of primary amino groups, but even at room temperature, N-trinitrophenylation of primary amines is concomitant with hydrolysis of the reagent. The production of picric acid, the product of TNBSA hydrolysis, lowers the sensitivity of the method. Heating accelerates hydrolysis more than condensation on amino groups. The optimal pH range is centered on the value 10. The optical density is generally evaluated at 340 or 420 nm. The former value corresponds to the maximal absorption of the final product, N-trinitrophenylamines. The latter value is relative to the Mesenheimer pi-complex, the well-known intermediate of the overall reaction. Both wavelengths are suitable for quantification of amines, but 420 nm seems to be the best. Further addition of sulfite is not necessary. The quantification of amines is somewhat hampered by compounds such as area and sodium dodecyl anions. The relative rates of reaction of diaminoacyl groups of protein with TNBSA differ depending on the substitution degree of the neighboring groups. Some of them do not react. The trinitrophenylation kinetic seems to be more dependent on protein structure than reactivity. In evaluation of the available lysine in glycated proteins, the distinction between reductive alkylation and Maillard-type condensation necessitates quantitative evaluation of nonalkylated lysine in protein hydrolysate, compared to results with the TNBSA method. Our results are confirmed by complete guanidization of the protein and subsequent determination of homoarginine in hydrolysates.