Neuroglobin A Preliminary Study Of Anti-free Radical Damage Mechanism

Free radicals are by-products of metabolism and lie in homeostasis of generating and scavenging in vivo, while this homeostasis mainly depends on the antioxidant system. It is well known that excessive free radicals in vivo can cause oxidative stress, and can react with biological macromolecules and leading to oxidative damage. At the same time, excessive free radicals can mediate various diseases, such as neurodegenerative diseases, stroke and cancer. Therefore, scavenging excessive free radicals has become an important way for prevention against these diseases. Neuroglobin (Ngb), the newly discovered third globin, was reported to be expressed mainly in nervous system by Burmester in 2000. It can bind oxygen with high affinity and can increase oxygen utilization in the brain. It has been reported that up-regulation of Ngb can protect neurons against ischemic/hypoxic insults. In recent years, several studies also suggested that Ngb may protect neuronal cells against oxidative damage, but most of these studies were carried out by using eukaryotic expression vectors, and provided that Ngb may protect neuronal cells against oxidative stress and have ROS scavenging effects in neuronal cells. However, there is no data on whether the Ngb protein itself can directly scavenge free radical and also no idea for the underlying mechanism. Therefore, the objective of this study is to address these points.First of all, we measured the antioxidant capability, scavenging free radicals, metal chelating capacity and the role against oxidative DNA damage using widely used the methods for determining free radical scavenging antioxidant activity in vitro and in cells. The antioxidant capacity of Ngb is a specific function because it is a member of oxygen-binding globin family. We measured the antioxidant and reducing power using ABTS and potassium ferricyanide reduction method in vitro, and found that rhNgb has the antioxidant capacity, although its antioxidant capacity is lower than Vc, GSH and NAC. However there is no reducing power for rhNgb, which may be due to the redox state of this protein.At the same time, we determined the scavenging activities of superoxide anion, hydrogen peroxide, hydroxyl radical because scavenging free radicals ability is quite important for antioxidants. We determined these activities using tetrazolium nitro blue-optical reduction, H2O2 inactivated enzyme, phenanthroline-Fe(II) assay respectively. Results showed that rhNgb had the scavenging capacities of superoxide anion, hydrogen peroxide, hydroxyl radicals. It was shown that rhNgb has the high capacity for superoxide anion scavenging, and has the same scavenging activity of Vc with little difference. The hydrogen peroxide scavenging activity of rhNgb was also strong, but its hydroxyl radical scavenging activity was lower than that of Vc and NAC. In addition, we also measured the peroxidase activity and scavenger DPPH free radical. Data showed that rhNgb almost has no peroxidase activity, and no DPPH scavenging activity.We also determined the Fe(II) chelating capacity and against DNA oxidative damage of rhNgb in order to better understanding the activities of rhNgb. Transition metal can catalyze the Fenton reaction and produce hydroxyl radical in the body, so the antioxidant ability of metal chelating capacity is a reflection of antioxidant. Result showed that rhNgb has some chelating ability for transition metal Fe(II), but is not complete dose-dependent. Its metal chelating capacity has dose-dependent at low concentration; its chelating ability begins to decline when rhNGB reaches the maximum concentration. This may be related to the iron porphyrin of rhNgb.To better understand the free radical scavenging activities of rhNgb, we detected the cellular cytotoxicity of rhNgb and scavenging excessive reactive oxygen species in neuronal PC12 cells. Although there are many methods for detecting intracellular ROS, we used DCFH-DA fluorescence probe because of the high sensitivity for excessive ROS in PC12 cells. Meanwhile, we measured the cytotoxicity of rhNGB using CCK-8 in PC12 cells to determine whether the activity of rhNgb for scavenging excessive ROS is related with the cytotoxicity or not. Our results showed that rhNgb has no cytotoxicity and could scavenge excessive intracellular ROS.In order to further study the mechanism of Ngb, it is suggested that His is a key amino acid in Ngb though the bioinformatics sequence analysis of Ngb protein, integrating with the functional conservation of amino acids. Then, it is showed that His in the Ngb protein plays a key role. Therefore, we constructed the mutant Ngb prokaryotic expression vector (pBV220-Ngb (H64V), pBV220-Ngb(H96A) and pBV220-Ngb(H64V/H96A)), and expressed in E. coli. Meanwhile, it was found that the bacteria expressing mutant Ngb shows the yellow color, while wild-type Ngb had red color. These results provided important information for further studying free radical scavenging mechanisms of Ngb. In summary, this paper conducted a series of studies for the antioxidant properties of rhNgb, and demonstrated that rhNgb is a potential antioxidant and has antioxidant capacity, scavenging free radicals activities, scavenging excessive intracellular ROS. At the same time, we found that rhNgb had metal chelating ability and protective effect on DNA damage induced by free radicals. The antioxidant capacity and free radical scavenging activity against oxidative damage may be the key point for the antioxidant properties of Ngb protein and will be further validated by using Ngb mutants. Our data have provided an experimental basis for further revealing neuroprotection function of Ngb and will advance the clinical application.