TNBSA (2,4,6-Trinitrobenzene sulfonic acid)

Thermo Scientific Pierce TNBSA Reagent is a 5% solution of trinitrobenzene sulfonic acid in methanol that reacts with primary amines (peptides or amino acids) to yield a soluble colored product, a property useful for various assay methods.

This Pierce Reagent solution contains trinitrobenzene sulfonic acid (TNBSA), which reacts readily with the primary amino groups of amino acids in aqueous solution at pH 8 to form yellow adducts. No colored derivatives are formed with the secondary amino acids praline and hydroxyproline. The colored derivatives are monitored at 335 to 345nm and have extinction coefficients in the range of 10,000-15,000. TNBSA has been used as a hydrophilic modifying reagent for the detection of primary amines in samples containing amino acids, peptides or proteins. It is an excellent reagent for rapid qualitative and quantitative estimation of these biomolecules.

Highlights:

• TNBSA component reacts readily with primary amino groups of amino acids in aqueous format at pH 8 to form yellow adducts
• Colored derivatives are monitored at 345nm and have extinction coefficients of 10,000 to 15,000
• Supplied as 5% solution (w/v) of TNBSA in methanol, which is easily diluted approximately 1000-fold in assay buffer for typical protein and peptide applications
• No colored derivatives are formed with secondary amino acids proline and hydroxyproline
• Applicable to solution or solid phase analysis

Properties of TNBSA:

• Alternative names: 2,4,6-trinitrobenzenesulfonic acid (TNBSA), picrylsulfonic acid, trinitrophenylsulfonic acid, trinitrobenzene sulfonate (TNBS)
• Chemical name: 2,4,6-trinitrobenzenesulfonic acid
• Reactive toward: Primary amines (–NH2)
• Chemical formula: C6H3N3O9S
• CAS number: 2508-19-2; 5400-70-4; 85600-65-3
• Molecular weight: 293.17

Product Details:

Chemical structure of 2,4,6-trinitrobenzene sulfonic acid (TNBSA). Thermo Scientific Pierce TNBSA Reagent is a 5% solution of this compound.

TNBSA reaction scheme for detection of primary amines. Trinitrobenzene sulfonate (TNBS) reacts with primary amines molecules to from an orange-colored product, whose absorbance at 335 to 345nm can be measured with a plate reader or spectrophotometer.

Example procedure for measuring protein primary amines (Hermanson, 2008, p. 127-128):

1. Dissolve or dialyze protein (20-200µg/mL) in 0.1M sodium bicarbonate buffer (pH 8.5). Avoid Tris or other amine-containing buffers.
2. Dilute supplied 5% TNBSA solution 500-fold in 0.1M sodium bicarbonate buffer (pH 8.5).
3. Add 0.5mL diluted TNBSA solution to 1mL of protein solution. Mix well.
4. Incubate at 37°C for 2 hours.
5. Add 0.5mL of 10% SDS and 0.25mL of 1N HCl to each sample to stop and stabilize reaction.
6. Measure absorbance of solution at 335nm. Determine concentration of primary amines by calculation from the extinction coefficient or by comparison to amino acid standards.

References:

1. Hermanson, G.T. (2008). Bioconjugate Techniques, Academic Press (Part No. 20036)
2. Goodwin, J.F. and Choi, S-Y. (1970) Quantification of protein solutions with trinitrobenzenesulfonic acid. Clinical Chemistry. 16, 24-31.
3. Snyder, S.L. and Sobocinski, P.Z. (1975) An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem. 64, 284-288.
4. Drozdovskaya, N.R., et al. (1982) FEBS Lett. 150, 385.
5. Takahashi, S., et al. (1984) Chem. Lett. (Jpn). 1, 127.