Study On The Interaction Of Heme Or Copper And Insulin Receptor Peptide And Its Catalized Insulin Receptor Peptide Nitration

Diabetes mellitus is one of the main diseases which threaten the health of the human being,the most common one is type 2 diabetes（T2D）,its key feature is insulin resistance.The atuophosphorylations of Tyr ¹¹⁵⁸,Tyr ¹¹⁶²,Tyr ¹163163 plays important role in insulin signal transduction,insulin resistance will happen if it is destroyed.Some research had discovered that the concentrations of heme and copper increased abnormally in T2D patients,while more protein nitration also had been detected,but the linkage between them remains unclear.Heme and Cu²⁺may cause nitration of tyrosine in the presence of reactive oxygen species and reactive nitrogen species.A truncated insulin receptor kinase domain fragment（1126-1165）（KK-1）,which contained three crucial autophosphorylation sites(Tyr¹¹⁵⁸,Tyr¹¹⁶² and Tyr¹¹⁶³)and favorable binding sites for heme,was used to explore molecular linkage of heme or Cu²⁺to insulin resistance.In this thesis,UV-vis absorbance spectra,fluorescence quenching and differential pulse voltammetry were used to explore KK-1/heme complex formation,TMB and ABTS were used as the substrate to compare the peroxidase activities of KK-1/heme and heme.Then fluorescence spectrometry was used to explore the production of dityrosine.Finally,dot blotting was used to explore the nitration of KK-1.Our results showed that heme bound to insulin receptor kinase domain peptide（1126-1165）at a stoichiometric ratio of 1:2.The binding amplified the peroxidative activity of heme itself and exacerbated the nitration of insulin receptor kinase domain in the presence of hydrogen peroxide and nitrite.Similarly,fluorescence quenching and differential pulse voltammetry were used to explore KK-1/Cu²⁺complex formation,BCA and coumarin were used to confirm the produtions of Cu⁺and·OH in the system of KK-1/Cu²⁺/H₂O₂ or Cu²⁺/H₂O₂.Then fluorescence spectrometry and dot blotting were used to explore the production of dityrosine and the nitration of KK-1 in the presence of KK-1/Cu²⁺,and the nitration impacts on its phosphorylation.·OH was produced more quickly in KK-1/Cu²⁺/H₂O₂,as a result,tyrosine is nitrated.We also found that nitration of Tyr¹¹⁵⁸ suppressed about 35%phosphorylation level of the key tyrosine residues.In conclusion,the disorder of heme or Cu²⁺will both nitrate Tyr ¹¹⁵⁸,Tyr ¹¹⁶²,Tyr ¹¹⁶³of KK-1 with nitrite and hydrogen peroxide,leading to the decrease of autophosphorylation,which will produce insulin resistance in the process of signal transduction following with type 2 diabetes.