Analytical Properties of Some Commercially Available Nitrate Reductase Enzymes Evaluated as Replacements for Cadmium in Automated, Semiautomated, and Manual Colorimetric Methods for Determination of Nitrate Plus Nitrite in Water.

摘要

A multiyear research effort at the U.S. Geological Survey (USGS) National Water Quality Laboratory (NWQL) evaluated several commercially available nitrate reductase (NaR) enzymes as replacements for toxic cadmium in longstanding automated colorimetric air-segmented continuous-flow analyzer (CFA) methods for determining nitrate plus nitrite (NOx) in water. This research culminated in USGS approved standard- and low-level enzymatic reduction, colorimetric automated discrete analyzer NOx methods that have been in routine operation at the NWQL since October 2011. The enzyme used in these methods (AtNaR2) is a product of recombinant expression of NaR from Arabidopsis thaliana (L.) Heynh. (mouseear cress) in the yeast Pichia pastoris. Because the scope of the validation report for these new automated discrete analyzer methods, published as U.S. Geological Survey Techniques and Methods 5B8, was limited to performance benchmarks and operational details, extensive foundational research with different enzymesprimarily YNaR1, a product of recombinant expression of NaR from Pichia angusta in the yeast Pichia pastorisremained unpublished until now. This report documents research and development at the NWQL that was foundational to development and validation of the discrete analyzer methods. It includes: (1) details of instrumentation used to acquire kinetics data for several NaR enzymes in the presence and absence of known or suspected inhibitors in relation to reaction temperature and reaction pH; and (2) validation resultsmethod detection limits, precision and bias estimates, spike recoveries, and interference studiesfor standard- and low-level automated colorimetric CFA-YNaR1 reduction NOx methods in relation to corresponding USGS approved CFA cadmium-reduction (CdR) NOx methods. The cornerstone of this validation is paired-sample statistical and graphical analysis of NOx concentrations.