TY - JOUR
T1 - Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase
AU - Woldman, Yakov Y.
AU - Sun, Jian
AU - Zweier, Jay L.
AU - Khramtsov, Valery V.
PY - 2009/11/15
Y1 - 2009/11/15
N2 - Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases subnanomolar, physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescence approach to direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphosphate to guanosine 3′,5′-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases by about 200 times when NO binds with sGC and, in so doing, provides a sensor for nitric oxide. Luminescence detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from the ATP-dependent luciferin-luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurement of NO generated by a nitric oxide donor (PAPA-NONOate), nitric oxide synthase, and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.
AB - Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases subnanomolar, physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescence approach to direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphosphate to guanosine 3′,5′-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases by about 200 times when NO binds with sGC and, in so doing, provides a sensor for nitric oxide. Luminescence detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from the ATP-dependent luciferin-luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurement of NO generated by a nitric oxide donor (PAPA-NONOate), nitric oxide synthase, and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.
KW - Chemiluminescence
KW - Free radicals
KW - Guanylyl cyclase
KW - Luciferase
KW - Nitric oxide
KW - Nitric oxide synthase
KW - NO donors
KW - Pyrophosphate
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U2 - 10.1016/j.freeradbiomed.2009.09.007
DO - 10.1016/j.freeradbiomed.2009.09.007
M3 - Article
C2 - 19751819
AN - SCOPUS:70350012271
SN - 0891-5849
VL - 47
SP - 1339
EP - 1345
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 10
ER -