Rapid spectrophotometric determination of 2,4,6-trinitrotoluene in a Pseudomonas enzyme assay

B. T. Oh, G. Sarath, P. J. Shea, R. A. Drijber, S. D. Comfort

Research output: Contribution to journalArticlepeer-review

25 Scopus citations


Although TNT (2,4,6-trinitrotoluene) and its degradation products can be quantified by HPLC, this method is not suitable for simultaneous analyses of the numerous samples typically encountered in enzyme studies. To solve this problem, we developed a simple and rapid spectrophotometric assay for TNT and tested the procedure using partially purified nitroreductase(s) from a Pseudomonas aeruginosa isolate, which transformed TNT in the culture medium. In highly alkaline solution, TNT (pK(a) = 11.99) exhibits significant absorbance at 447 nm, while major metabolites, 2-amino-4,6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and 2,6-diamino-4-nitrotoluene (2,6DANT) display no absorbance at this wavelength. Assay mixtures of TNT, Tris-HCl buffer, a reductant, and the enzyme(s) were analyzed by measuring absorbance 4 min after adjusting the pH to 12.2. TNT transformation to colorless metabolites was linear with respect to protein and substrate concentrations. Using the assay, we determined that TNT nitroreductase(s) from the isolate required an electron donor and preferred NADH to NADPH. TNT transformation increased when NAD was recycled to NADH using glucose-6- phosphate (GP) and glucose-6-phosphate dehydrogenase (GPDH). Enzymatic transformation of TNT was completely inhibited by Cu2+ (5 mM) and was partially inhibited by other divalent metallic cations. Because the assay is sensitive to ammonium sulfate, dithiothreitol, ascorbic acid, and sodium phosphate, extracts should be assayed in the absence of these components. (C) 2000 Published by Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)149-158
Number of pages10
JournalJournal of Microbiological Methods
Issue number2
StatePublished - Oct 2000


  • Spectrophotometric assay
  • TNT
  • TNT nitroreductase(s)

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)


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