GC-MS determination of nitrous anhydrase activity of bovine and human carbonic anhydrase II and IV

The most widely recognized activity of the large family of the metalloenzyme carbonic anhydrases (CAs) is the diffusion-controlled hydration of CO₂ to HCO₃^- and one proton, and the less rapid dehydration of HCO₃^- to CO₂: CO₂ + H₂O ⇆ HCO₃^- + H⁺. CAs also catalyze the reaction of water with other electrophiles such as aromatic esters, sulfates and phosphates, thus contributing to lending to CAs esterase, sulfatase and phosphatase activity, respectively. Renal CAII and CAIV are involved in the reabsorption of nitrite, the autoxidation product of the signalling molecule nitric oxide (NO): 4 NO + O₂ + 2 H₂O → 4 ONO^- + 4 H⁺. Bovine and human CAII and CAIV have been reported to exert nitrite reductase and nitrous anhydride activity: 2 NO₂^- + 2 H⁺ ⇆ [2 HONO] ⇆ N₂O₃ + H₂O. In the presence of L-cysteine, NO may be formed. In the literature, these issues are controversial, mainly due to analytical shortcomings, i.e., the inability to detect authentic HONO and N₂O₃. Here, we present a gas chromatography-mass spectrometry (GC-MS) assay to unambiguously detect and quantify the nitrous anhydrase activity of CAs. The assay is based on the hydrolysis of N₂O₃ in H₂¹⁸O to form ON¹⁸O^- and ¹⁸ON¹⁸O^-. After pentafluorobenzyl bromide derivatization and electron capture negative-ion chemical ionization of the pentafluorobenzyl nitro derivatives, quantification is performed by selected-ion monitoring of the anions with mass-to-charge (m/z) ratios of 46 (ONO^-), m/z 48 (ON¹⁸O^- and ¹⁸ONO^-), m/z 50 (¹⁸ON¹⁸O^-) and m/z 47 (O¹⁵NO^-, internal standard).