Clinical Chemistry, Vol 16, 24-31, Copyright © 1970 by the American Association for Clinical Chemistry

Quantification of Protein Solutions with Trinitrobenzenesulfonic Acid

¹ Department of Biochemistry and the General Clinical Research Center for Children, Wayne State University School of Medicine, and the Children’s Hospital of Michigan, Detroit, Mich. 48202.

Trinitrobenzenesulfonic acid (TNBS) may be used for quantifying dilute solutions of protein. The trinitrophenyl protein—sulfite complexes resulting from reaction of TNBS with protein in the presence of sulfite are highly colored and exhibit maximum spectral absorbance at 420 nm. The TNBS protein—sulfite complex is formed by a simple two-stage condensation and complexation reaction. Protein in concentrations as low as 20 µg may be detected by the reaction. The method devised for protein quantification compares favorably with an ultraviolet spectrophotometric method and the conventional biuret procedure. Bilirubin and hemoglobin interference with the TNBS method is negligible. The procedure is useful for measuring total protein and globulin in serum and cerebrospinal fluid and also for measuring minor serum protein components. Procedural parameters and factors affecting the measurement of protein by the TNBS—sulfite reaction are presented.