Development and validation of a novel DTNB-based assay for tryptophanase activity: A tool for research applications

Tryptophanase (TnaA) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the α, β-elimination reaction, producing indole, pyruvate, and ammonia, thereby linking carbon and nitrogen metabolism. TnaA enables tryptophan to serve as a source of carbon, nitrogen, and energy. TnaA also converts S-methyl-L-cysteine (SMC) to methyl mercaptan, pyruvate, and ammonia with comparable efficiency, although its usual physiological substrate is tryptophan. TnaA is primarily found in bacteria and has many biological functions. Its roles in metabolism and bacterial activity, as well as its particularly significant medicinal applications, have garnered considerable interest in recent years. This article introduces a new spectrophotometric method for detecting TnaA activity. In this method, SMC is used as the substrate, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is the reagent. Existing methods for quantifying TnaA activity generally rely on the indirect detection of indole or pyruvate. TnaA cleaves SMC to produce methyl mercaptan, which reduces DTNB to form a yellow chromophore, 5-thio-2-nitrobenzoate (TNB^-), with absorbance around 412 nm. The optimal pH for this reaction was 7.8 in dihydrogen phosphate/monohydrogen phosphate buffer, with 5'-phosphate pyridoxal as the cofactor. The current methodology was validated for linearity within the range of 5-1000 U/L, accuracy with a relative standard deviation (RSD) ≤ 5%, and a detection limit of 3.47 U/L. To our knowledge, this is the first report demonstrating that SMC serves as an analytically useful substrate for measuring DTNB-based TnaA activity, offering a direct, solvent-free alternative to established indole-based assays. The DTNB-SMC method provides a sensitive, cost-effective, and high-throughput-compatible tool for TnaA research in microbiology, metabolomics, and drug discovery.