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

B. T. Oh, G. Sarath, Patrick J Shea, R. A. Drijber, Steven Comfort

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

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 Cu 2+ (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
Volume42
Issue number2
DOIs
StatePublished - Oct 1 2000

Fingerprint

Trinitrotoluene
Enzyme Assays
Pseudomonas
Nitroreductases
NAD
Glucose-6-Phosphate
Tromethamine
Glucosephosphate Dehydrogenase
Dithiothreitol
Reducing Agents
Divalent Cations
Ammonium Sulfate
Enzymes
NADP
Pseudomonas aeruginosa
Ascorbic Acid
Culture Media
High Pressure Liquid Chromatography
Electrons

Keywords

  • Spectrophotometric assay
  • TNT
  • TNT nitroreductase(s)

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

Cite this

Rapid spectrophotometric determination of 2,4,6-trinitrotoluene in a Pseudomonas enzyme assay. / Oh, B. T.; Sarath, G.; Shea, Patrick J; Drijber, R. A.; Comfort, Steven.

In: Journal of Microbiological Methods, Vol. 42, No. 2, 01.10.2000, p. 149-158.

Research output: Contribution to journalArticle

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