Study On The Interaction Of FeTPPS With Serum Albumin And Human Islet Amyloid Polypeptide

5,10,15,20-Tetrakis?4-sulfonatophenyl?porphyrinato iron?III?chloride?FeTPPS?is widely used as a scavenger of peroxynitrite in many studies,which has a protective effect against the damage induced by endogenic and ectogenic peroxynitrite.However,FeTPPS is the water-soluble derivative of heme and has the same porphyrin ring and iron center as heme.Thus,FeTPPS should possess the similar biological effect as heme.In fact,heme exerts a dual role in vivo.As the prosthetic group of heme proteins,heme plays an essential role in various biological reactions.Whereas in excessive amounts,free heme can catalyze the production of free radicals through Fenton chemistry and cause severe damage.In the presence of H₂O₂ and NaNO₂,heme also catalyzes protein tyrosine nitration.However,up to now,no studies have found that FeTPPS exerted obvious damage on cells and tissue,which is much different from heme.Therefore,the research on the harmlessness of FeTPPS to cells and tissue is important to understand the safety of FeTPPS as a therapeutic agent.Besides,since both reactive nitrogen species?RNS?and human islet amyloid polypeptide?hIAPP?aggregation play important roles in the pathogenesis of type II diabetes,it is necessary to study whether FeTPPS,a peroxynitrite scavenger and heme analogue,could inhibit hIAPP aggregation like heme.The study will provide the theoretical support for using FeTPPS as a potential dual-functional drug in the prevention and treatment of type II diabetes mellitus which could both scavenge peroxynitrite and inhibit the toxicity of hIAPP.To answer the above questions,this article studied FeTPPS in the following three areas.?1?The ability of FeTPPS in catalyzing protein nitration of living cells and its effect on cell viability.Dot blotting result demonstrated that FeTPPS could induce a certain extent of protein tyrosine nitration in human hepatocellular carcinoma cell?HepG2?in the presence of H₂O₂ and NaNO₂.However,the cell viability assay showed FeTPPS could effectively reduce H₂O₂-induced cells injury.Western blotting result revealed FeTPPS could not up-regulate heme oxygenase-1?HO-1?,which meant the protection ability of FeTPPS was not related to HO-1.?2?The binding properties of bovine serum albumin?BSA?and FeTPPS and the direct inhibition of which on the pro-oxidative activity of FeTPPS.Fluorescence quenching study showed BSA could bind to FeTPPS with high affinity(K_b¹0⁹ M^-1),and synchronous fluorescence quenching spectroscopic study further demonstrated that binding with FeTPPS could increase the polarity and weaken the hydrophobicity of the tyrosine residues in BSA.Molecular docking manifested the binding site of FeTPPS was at the surface region of BSA.It was found that the binding of BSA could effectively inhibit the pro-oxidative activity of FeTPPS and form a low reactivity of the BSA-FeTPPS complex.In addition,BSA could protect FeTPPS against oxidative degradation which could prevent the release of iron.These findings may partly explain the reason why FeTPPS could be used safely in vivo.?3?The inhibition of FeTPPS on hIAPP amyloid aggregation and the effect of which on hIAPP-induced cell damage.UV-Vis spectroscopy showed FeTPPS could bind to hIAPP monomer.The results of fluorescence assay,transmission electron microscopy?TEM?and NuPAGE^?gel electrophoresis demonstrated FeTPPS could effectively inhibit the amyloid aggregation of hIAPP and the iron center of FeTPPS play a role in the inhibition.In addition,the studies of this paper also indicated FeTPPS could partly dismantle the mature fibre of hIAPP.The MTT assay revealed the strong inhibition of FeTPPS on the cytotoxicity induced by amyloid aggregation of hIAPP,in which the iron center of FeTPPS played a key role.The DCFH-DA assay indicated that FeTPPS could inhibit oxidative stress induced by the aggregation of hIAPP.This study may provide a guidance for designing the new anti-aggregation medicines for hIAPP based on the metal porphyrin.