Development and evaluation of a bi-enzymatic nitric oxide reduction system

Abstract

Nitric oxide is a small diatomic molecule and is part of the nitrogen radical species. As a gas, it diffuses easily across cell membranes and is involved in numerous physiological processes and inflammation. This peculiar molecule has a dual role in inflammation. NO is one of the first signals to commence an innate immune response and it is involved in the resolution of inflammation. In the control of inflammation it is crucial to resolve NO bursts to promote tissue healing. The failure thereof results in the progression of inflammation with potentially catastrophic consequences for the host. This study aimed to develop a nitric oxide reduction system as a research tool which could facilitate the understanding of the intricate role of NO in inflammation. Numerous chemical tools have been used to study NO biology, but were found to interfere in other metabolic pathways; thereby masking the role of NO. The nitric oxide reduction system entails the reduction of NO by nitric oxide reductase (NOR) with the concomitant oxidation of glucose by glucose dehydrogenase (GDH). The latter enzyme recycles the cofactor NADH in such a way that NO is continuously reduced. This bi-enzymatic cofactor recycling system presents the advantage of NO removal without any interference of metabolic pathways. Here, we propose that the continuous reduction of NO by the NOR system could be used to elucidate the role of NO in an innate immune response. The construction of the NOR system commenced with development of fast and reliable spectrophotometric NADH-enzyme activity assay. This assay was essential for the quantification of enzyme activity and was used throughout the study for the purification of NOR, characterisation of NOR as well as the determination of enzyme activity maintenance after enzyme immobilisation. Both enzymes were immobilised onto five carriers with two different functional group chemistries and three functional group densities. The carboxyl functionalised carrier with the lowest functional group density was the most suitable immobilisation carrier by maintaining the highest enzyme activity for NOR and GDH. Upon co-immobilisation of both enzymes, an average of 0.088 μmoles NADH.min-1 for NOR and 0.077 μmoles NAD.min-1 for GDH cofactor oxidation rate was achieved. Furthermore, the cofactor was recycled six times with the concomitant consumption of the enzymes’ substrates. Subsequently, the NOR system was evaluated for its potential as a research tool in an in vitro inflammation model. The continuous reduction of NO was established which highlights the NOR system suitability as a research tool. However, its evaluation as a potential anti-inflammatory reagent indicated that the chosen carrier has immunogenic properties of its own. The inflammation response elicited by this carrier alone was in part abrogated by the immobilisation of enzymes in the eventual NOR system assembly, thereby providing a scope for future work and further optimisation of this anti-inflammatory reagent.