Colorimetric Determination of Nitrate Plus Nitrite in Water by Enzymatic Reduction, Automated Discrete Analyzer Methods

This report documents work at the U.S. Geological Sur- vey (USGS) National Water Quality Laboratory (NWQL) to validate enzymatic reduction, colorimetric determinative meth- ods for nitrate + nitrite in filtered water by automated discrete analysis. In these standard- and low-level methods (USGS I-2547-11 and I-2548-11), nitrate is reduced to nitrite with nontoxic, soluble nitrate reductase rather than toxic, granular, copperized cadmium used in the longstanding USGS auto- mated continuous-flow analyzer methods I-2545-90 (NWQL laboratory code 1975) and I-2546-91 (NWQL laboratory code 1979). Colorimetric reagents used to determine resulting nitrite in aforementioned enzymatic- and cadmium-reduction meth- ods are identical. The enzyme used in these discrete analyzer methods, designated AtNaR2 by its manufacturer, is produced by recombinant expression of the nitrate reductase gene from wall cress (Arabidopsis thaliana) in the yeast Pichia pastoris. Unlike other commercially available nitrate reductases we evaluated, AtNaR2 maintains high activity at 37 C and is not inhibited by high-phenolic-content humic acids at reaction temperatures in the range of 20 C to 37 C. These previously unrecognized AtNaR2 characteristics are essential for success- ful performance of discrete analyzer nitrate + nitrite assays (henceforth, DA-AtNaR2) described here.