Design and development of spectrophotometric enzymatic cyanide assays

Determination of free cyanide (fCN) is required for various industrial, environmental, food, and clinical samples. Enzymatic methods are not widely used in this field despite their selectivity and mild conditions. Therefore, we present here a proof of concept for new spectrophotometric enzymatic assays of fCN. These are based on the hydrolysis of fCN affording the readily detectable NADH. fCN is hydrolyzed either in one step by cyanide dihydratase (CynD) or in two steps by cyanide hydratase (CynH) and formamidase (AmiF). An advantage of the latter route is the higher activity of CynH and AmiF compared to CynD. In both cases, the resulting formate is then transformed by an NAD-dependent formate dehydrogenase (FDH). The NADH thus formed is quantified colorimetrically using a known method based on a reduction of a tetrazolium salt (WST-8) with NADH. The developed assays of fCN are selective except for formic acid interference, proceed under mild conditions, and, moreover, fCN is detoxified during the reactions. The assays proceeded in a microtiter plate format. The limit of detection (LOD) and the limit of quantification (LOQ) were lower for the three-enzyme (CynH-AmiF-FDH) method (7.00 and 21.2 µmol/L, respectively) than for the two-enzyme (CynD-FDH) method (10.7 and 32.4 µmol/L, respectively). In conclusion, the new fCN assays presented in this work are selective, high-throughput, do not require harsh conditions, and use only small amounts of chemicals and enzymes.