Eliminating absorbing interference using the H-point standard addition method: case of Griess assay in the presence of interferent heme enzymes such as NOS.
Standard calibration methods used to determine trace analytes usually yield significant deviations from the actual analyte value in the presence of interferents in the assay media. These deviations become of particular concern when the concentration of the analyte is low, and when the results are used to draw mechanistic or kinetic conclusions, for instance in enzyme structure-function studies. In these circumstances, the H-point standard addition method (HPSAM) provides superior precision and accuracy. This method is developed here for the case of the spectrophotometric Griess assay used to determine nitrite in various enzymology investigations, such as nitrite determination in studies of nitrite reductases (NiR), or when determining nitrite as a breakdown product of nitric oxide synthesized by NOS enzymes. The results obtained by HPSAM are contrasted with those of the traditional calibration method.
Indika
Perera N
PN
Chemistry Department, Cleveland State University, SR 397, Cleveland, OH 44115, USA.
Bayachou
Mekki
M
D000042:Absorption; D000818:Animals; D002138:Calibration; D005029:Ethylenediamines; D016166:Free Radical Scavengers / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D009211:Myoglobin / Q000737:chemistry / Q000592:standards; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000592:standards; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods; D013424:Sulfanilamides
D005029:Ethylenediamines; D016166:Free Radical Scavengers; C095000:Griess reagent; D009211:Myoglobin; D009573:Nitrites; D013424:Sulfanilamides; D006418:Heme; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse