Ultrafast Excited State Dynamics Of Bile Pigments In Proteins

Bile pigments(biliverdin and bilirubin)are a series of linear tetrapyrrole molecules in organisms,and often exist in the form of binding with proteins.Among them,biliverdin(BV)is indispensable in the photosynthesis of plants and algae.Bilirubin(BR)is an important metabolite in animals,and its photochemical products are closely related to the clinical phototherapy effect of neonatal jaundice(hyperbilirubinemia).Due to its flexibility structure,bile pigments can show obvious photothermal/photoacoustic effects,excited state proton transfer,photo-induced conformational change and strong fluorescence emission in different protein cavities.Therefore,functional proteins with BV as chromophore were reported to show great application potential in the fields of imaging(especially depth imaging)and photothermal therapy in recent years.Meanwhile,clinical data suggest that the photochemical reaction efficiency of BR is closely related to the human serum albumin(HSA)environment and irradiation wavelength.However,due to the structural variability of bile pigment and the complexity of protein cavity microenvironment,the excited state dynamics of bile pigments has not been fully elucidated,which hinders the further development and application of these moeleculs.In order to better understand the influence of protein cavity on the photophysicochemistry properties of bile pigments,we studied the excited state dynamics of biliverdin and its derivatives in solution and different protein environments by using ultrafast time-resolved spectroscopy.Our data reveal the relationship between the environment and the excited state relaxation process of biliverdin and provide theoretical foundation for the application of functional protein based on biliverdin in the field of fluorescence and photoacoustic imaging.Besides,we also studied the excited state dynamics of BR-HSA complex by adjusting fatty acid concentration in the protein environment(internal factors)and the irradiation wavelength(external factors).The influence of external environment on the photochemical reaction of BR was demonstrated for the first time.Based on our results,we propose a new irradiation scheme for hyperbilirubinemia phototherapy and this may lead to a new breakthrough in the related clinical medical research.The main research in this dissertation are as follows:1.The characteristic spectral properties and complete excited state dynamics of biliverdin dimethyl ester in solution were studied by using steady-state and ultrafast time-resolved spectroscopy.The influence of external environment on the excited state decay pathways was elucidated,and the formation of a new species with fluorescence quantum yield enhancement of 37 times was confirmed.2.The excited state dynamics of biliverdlin in sm URFP and its mutants was studied by using various spectroscopic techniques.The ground state heterogeneity in the system was confirmed,and its influence on the excited state dynamics was explained.From a new perspective,the reasons why smURFP has higher fluorescence quantum yield than other near-infrared fluorescent proteins were clarified.3.Using various spectral techniques,a complete photochemical reaction model of BR in HSA was established,and the importance of fatty acids to regulate the reaction was clarified.The observed results in clinical field were explained from the mechanism.4.The relationship between the formation rate of bilirubin photoreaction products and the irradiation condition is revealed by using the pump-repump-probe spectroogy technique.A new irradiation scheme which is beneficial to the results of phototherapy is proposed.This work explains the observation results in the field of clinical medicine from the mechanism,and provides theoretical support and new ideas for the exploration of better irradiation scheme to improve phototherapy of jaundice.