Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60degreesC, until nitrite reductase activity was less than or equal to25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 muM) were incubated with the nitrate reductase preparation for 3 h at 37degreesC, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods. (C) 2004 Elsevier Inc. All rights reserved.