Spectral Properties Of Bilirubin And Its Analogue Interacting With Metal Ions

Bilirubin?BR?is one of the metabolites of heme in mammals,mainly distributed in blood and bile.When the level of BR in the body is too high,excess BR may interact with metal ions,leading to a variety of diseases,such as hyperbilirubinemia and gallstones.Therefore,studying the interaction of BR with metal ions is of great significance for the treatment of these diseases.At the same time,as a linear tetrapyrrole molecule,the luminescence property of BR is also worth studying.The influence of metal ions on fluorescence intensity of BR contributes to the design and improvement of bioprobes.Firstly,UV-visible absorption and steady-state fluorescence spectroscopy were used to study the interaction of bilirubin with metal ions such as Zn²⁺,Cu²⁺,Fe³⁺,Al³⁺,Ni²⁺and Mg²⁺.Among them,?1?Zn²⁺has a fluorescence enhancement to BR and a new fluorescence emission peak appears;?2?BR has an obvious interaction with Cu²⁺,Fe³⁺,Al³⁺,and the absorption peak of BR is blue-shifted.The interaction between Cu²⁺and BR produces a new absorption peak at the red end.These metal ions all weaken the fluorescence intensity of BR;?3?There is no significant change in the shape and intensity of spectrum of BR after mixing with Ni²⁺and Mg²⁺in solution,indicating that these two metal ions hardly interact with BR.Since BR is usually present in the form of binding to serum albumin,and Cu²⁺is a common metal toxic substance,the effects of Cu²⁺on BR-BSA complex were studied by UV-visible absorption,steady-state fluorescence spectroscopy,time-correlated single photon counting and transient absorption spectroscopy.Steady-state fluorescence spectroscopy showed that the fluorescence was attenuated after the addition of Cu²⁺in the BR-BSA complex solution,but it was still significantly enhanced compared with BR.Transient absorption spectroscopy indicated a rapid charge transfer between the Cu²⁺and BR-BSA complex.Then we studied the interaction of the bilirubin analog,bilirubin dimethyl ester?BRE?,with various metal ions.The coordination point of BRE with metal is less than BR,and the complexity of the product is reduced,which is helpful for spectral analysis.?1?The excited-state spectral characteristics of BRE and Cu²⁺complex were studied by the combination of visible and infrared-band femtosecond absorption spectroscopy.We have proposed and confirmed the reason for the fluorescence quenching of BRE is the ligand-to-metal charge transfer?LMCT?.Compared with the spectral properties of BRE,this charge transfer state provides a non-radiative de-excitation pathway to the BRE-Cu²⁺complex,reducing the fluorescence quantum yield of BRE to⁵%of its original value.Two lifetimes?11 ps and 186 ps?were obtained by fitting the kinetics of the excited state spectra of the complex.We assign the 11 ps component to the build-up process of the LMCT state,while the 186 ps is the lifetime of the charge transfer state.?2?Then,the fluorescence enhancement phenomenon of Zn²⁺to BRE was studied.The combination of BRE and Zn²⁺produces a fluorophore with a fluorescence lifetime of nanoseconds.The fixation of Zn²⁺to BRE reduces the energy loss caused by non-radiative transition and enhances fluorescence.?3?Moreover,some interesting phenomenon between BRE and other metal ions such as Co²⁺?Ni²⁺and Mn²⁺,and the mechanism remains to be further studied.