[An enzymatic sensor for estimation of the content of nitro and nitroso compounds in biological objects]

Nitric oxide (NO) generation in the biological cells and tissues is a cause of the production of some nitro and nitroso compounds that differ in their physiological significance and toxicity. The resultant problem in the estimation of the content of all nitro and nitroso compounds totally and singly in complex systems, such as biological objects, remains to be very urgent since simple, highly sensitive and highly selective methods have not been proposed so far to determine all these NO derivatives. The method based on some specific biochemical property of NO metabolites, which manifests itself under physiological conditions and permits the fixation of all nitro and nitroso compounds without their prior modification fraught with unpredictable artifacts, seems to be optimal. The authors have designed an enzymatic sensor based on the previously established ability to inhibit the enzyme catalase in the presence of halide ions. The level of dinitrosyl iron complex (DNIC) was measured, by using its property to lose its ability to inhibit catalase on addition of a trap of NO and an iron chelator to a reaction medium. S-nitrosothiols are detected as substances that are able to produce DNIC after addition of ferrous iron and thiols, unlike nitrite that has not this property. The total content of nitro compounds was estimated, by reducing to nitroso compounds by vanadium (III) chloride. The nitro compounds showing the properties of NO donors (RNO2) were determined as the substances that acquire the properties of DNIC in the presence of ferrous iron and thiols. The content of nitrates was estimated as a difference between the total level of nitro compounds and the content of RNO2. The sensitivity of this method was as high as 50 nM. That is it is more than an order higher in sensitivity than the classical methods based on the Griess reaction. By keeping its high sensitivity in mind, the proposed catalase method as an enzymatic detector of nitro and nitroso compounds allows one to detect these compounds in the neutral medium, without pre-purifying the object, thereby preventing the influence of the factors that contribute to the uncontrolled modification of the compounds under study.