Discovery and microassay of a nitrite-dependent carbonic anhydrase activity by stable-isotope dilution gas chromatography-mass spectrometry

Amino Acids
Maximilian ZinkeDimitrios Tsikas

Abstract

The intrinsic activity of carbonic anhydrase (CA) is the hydration of CO2 to carbonic acid and its dehydration to CO2. CA may also function as esterase and phosphatase. Recently, we demonstrated that renal CA is mainly responsible for the reabsorption of nitrite (NO2(-)) which is the most abundant reservoir of the biologically highly potent nitric oxide (NO). By means of a stable-isotope dilution GC-MS method, we discovered a novel CA activity which strictly depends upon nitrite. We found that bovine erythrocytic CAII (beCAII) catalyses the incorporation of (18)O from H2 (18)O into nitrite at pH 7.4. After derivatization with pentafluorobenzyl bromide, gas chromatographic separation and mass spectrometric analysis, we detected ions at m/z 48 for singly (18)O-labelled nitrite ((16)O=N-(18)O(-)/(18)O=N-(16)O(-)) and at m/z 50 for doubly (18)O-labelled nitrite ((18)O=N-(18)O(-)) in addition to m/z 46 for unlabelled nitrite. Using (15)N-labelled nitrite ((15)NO2 (-), m/z 47) as an internal standard and selected-ion monitoring of m/z 46, m/z 48, m/z 50 and m/z 47, we developed a GC-MS microassay for the quantitative determination of the nitrite-dependent beCAII activity. The CA inhibitors acetazolamide and FC5 207A did not alter beC...Continue Reading

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Citations

Oct 27, 2015·Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology·Daniel Carrie, Kathleen M Gilmour
Aug 27, 2016·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Dimitrios Tsikas
Sep 4, 2018·Expert Opinion on Therapeutic Patents·Claudiu T Supuran
Jul 28, 2018·BioMed Research International·Maciej JakubowskiAdrian Doroszko
Oct 29, 2019·British Journal of Pharmacology·Ling WangMark T Gladwin
Jan 30, 2021·Journal of Enzyme Inhibition and Medicinal Chemistry·Dimitrios Tsikas, Stepan Gambaryan

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