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Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were inactivated by incubation with nitric oxide under anaerobic conditions in the presence of xanthine or allopurinol. The inactivation was not pronounced in the absence of an electron donor, indicating that only the reduced enzyme form was inactivated by nitric oxide. The second-order rate constant of the reaction between reduced XO and nitric oxide was determined to be 14.8 +/- 1.4 M-1 s-1 at 25 degrees C. The inactivated enzymes lacked xanthine-dichlorophenolindophenol activity, and the oxypurinol-bound form of XO was partly protected from the inactivation. The absorption spectrum of the inactivated enzyme was not markedly different from that of the normal enzyme. The flavin and iron-sulfur centers of inactivated XO were reduced by dithionite and reoxidized readily with oxygen, and inactivated XDH retained electron transfer activities from NADH to electron acceptors, consistent with the conclusion that the flavin and iron-sulfur centers of the inactivated enzyme both remained intact. Inactivated XO reduced with 6-methylpurine showed no "very rapid" spectra, indicating that the molybdopterin moiety was damaged. Furthermore, inactivated XO reduced by dithionite showed the same slow Mo(V) spectrum as that derived from the desulfo-type enzyme. On the other hand, inactivated XO reduced by dithionite exhibited the same signals for iron-sulfur centers as the normal enzyme. Inactivated XO recovered its activity in the presence of a sulfide-generating system. It is concluded that nitric oxide reacts with an essential sulfur of the reduced molybdenum center of XO and XDH to produce desulfo-type inactive enzymes.

The effects of nitric oxide (NO) on xanthine oxidase (XOD) activity and the site(s) of the redox center(s) affected were investigated. XOD activity was determined by superoxide (O2-) generation and uric acid formation. NO reversibly and dose-dependently suppressed XOD activity in both determination methods. The suppression interval also disclosed a dose-dependent prolongation. The suppression occurred irrespective of the presence or absence of xanthine; indicating that the reaction product of NO and O2-, peroxynitrite, is not responsible for the suppression. Application of synthesized peroxynitrite did not affect XOD activity up to 2 microM. Methylene blue, which is an electron acceptor from Fe/S center, prevented the NO-induced inactivation. The results indicate that NO suppresses XOD activity through reversible alteration of the flavin prosthetic site.

Recently, 5,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) has been applied to detect biological free radicals. However, DBNBS has various non-specific reactions which lead to preplexing results. Thus, we investigated some basic reactions of DBNBS in combination of other nitroso spin traps to assign DBNBS spin adducts derived from human platelets which presumably related to the endothelium-derived relaxing factor (EDRF). The collagen activated platelets yielded four spin adducts (ST, LT, SS, and LS) in the presence of DBNBS (40 mM). The broad triplet due to ST was also observed by bubbling NO gas into a DBNBS solution. To identify ST, nitrosobenzene (NB) in dry dioxane was mixed with NO-saturated dioxane. The NB-NO spin adduct was observed but decomposed into diphenyl aminoxyl by the addition of H2O indicating that the primary adduct formed by the reaction of NO and DBNBS is unstable and turns into a dimerization product. Although ST could be eliminated by the inhibitor of EDRF, ST was shown to be produced by non-specific reactions. Another triplet was assigned to an S-centered radical because thiyl radicals which were generated from either the decomposition of S-nitrosothiol, or glutathione oxidation exhibited almost identical triplet signals. The other two sextets were assigned to C-centered radical adducts. Thus, DBNBS detected NQ-related, S-centered, and two C-centered radicals derived from human platelets. Special cautions are necessary for the identification of DBNBS spin adducts in a biological system to exclude artifactual radicals.

An NO-selective electrode was developed as an easily applicable tool for a real-time nitric oxide (NO) measurement. The working electrode (0.2 mm diam) was made from Pt/Ir alloy coated with a three-layered membrane. The counterelectrode was made from a carbon fiber. When a stable NO donor, S-nitroso-N-acetyl-dl-penicillamine, was applied, the electrode current increased in a dose-dependent fashion. The current and calculated NO concentration showed a linear relationship in the range from 0.2 nM (S/N = 1) to 1 µM of NO. The response of the electrode was 1.14 ± 0.09 s. The effects of temperature, pH, and chemicals other than NO on the electrode current were also evaluated. Electrodes which were placed in the luminal side of rat aortic rings exhibited 30 pA of current due to NO generation induced by the addition of 10[sup -6] M of acetylcholine. The current was eliminated in the presence of 50 µM N[sup G]-monomethyl-L-arginine, an inhibitor of NO synthase. Thus, this NO-selective electrode is applicable to real-time NO assay in biological systems. [ABSTRACT FROM AUTHOR]