The Effect Of Dopamine Quinone On The Activity Of LDHB And ENO1 Protein

Purpose:As a common neurodegenerative disease,pathogenesis of Parkinson’s disease is still unclear.We propose that,in neurons,dopamine quinone which is a production of dopamine oxidation,binds and modifies protein,causes disfunction of protein and cell death in the end.In this paper,we purified LDHB and ENO1 protein in vitro,then investigated effect of dopamine quinone on enzyme protein by treated protein sample with dopamine in different condition,and explored the relationship between toxicity of dopamine quinone and pathogenesis of Parkinson’s disease further.Methods:The gene of LDHB and ENO1 protein was amplified by PCR,then was ligated to expression vector(pProEX-HTb).The recombinant was transformed into the BL21 bacterial competent.Bacteria culture was induced to express LDHB and ENO1 protein by IPTG(Isopropyl β-D-Thiogalactoside).The bacteria was disrupted by ultrasound.The protein was purified via Ni-NTA affinity chromatography.Enzyme activity was determinated by change of absorbance after substrate of enzyme was added in protein sample.Before enzyme activity of protein sample was determinated,the protein sample was incubated with dopamine in different conditions.Denatured polyacrylamide gel electrophoresis was performed for each protein sample after incubation.Protein sample were analysed by Coomassie dye and NBT/glycinate redox-cycling staining.Finally,western blots was performed to analysis of LDHB protein sample.Results:The gene sequence of LDHB and ENO1 protein,which was amplified by PCR,was correct.The purified protein possessed biological enzyme activity.After LDHB and ENO1 protein sample was treated with the same concentration of dopamine for different time,the loss of LDHB and ENO1 protein activity was increased when treatment time was extended.After protein sample was incubated with different concentration of dopamine for same time,the loss of two protein activity increased when concentration of dopamine was increasd.The catalytic activity of protein was decreased when concentration of dopamine was increasd.If glutathione was added in sample in advance,it stopped the loss of activity and protected the protein activity.The dopamine quinone,which was catalysed by tyrosinase that converted dopamine into dopamine quinone without reactive oxygen species,could bind protein and decreased the activity of protein.When glutathione was added in sample in advance,the loss of activity was disappeared.Results of polyacrylamide gel electrophoresis showed the protein to aggregate into polymer after incubated with dopamine.The quantity of ploymer in proportion to time of treatment and concentration of dopamine.Results of NBT/glycinate redox-cycling staining showed that the polymer was caused by dopamine quinone,while glutathione stopped the aggregations of proteins.In the end,results of western blots showed the polymer is the dopamine quinone binding protein in LDHB protein sample,which was treated by dopamine.Conclution:The purified LDHB and ENO1 protein in vitro possessed biological enzyme activity in considerable degree.After LDHB and ENO1 protein were incubated with dopamine,dopamine quinone which was the production of DA oxidation,bound and modified protein.The protein formed into polymer,and activity of protein was decreased.The effect was more serious when the treatment time was longer or the concentration of dopamine was higher.