Study On The Comparision Of Structures And Properties Between Chicken Egg Yolk And White Riboflavin Binding Protein

Riboflavin binding protein?RBP?,one of the most important proteins in eggs,exists in both egg white and egg yolk.It binds to riboflavin and plays an important role in embryonic development.However,the current research on the structural properties of egg white and the riboflavin-binding protein is still incomplete.In this paper,the egg white-and egg yolk-riboflavin-binding protein were identified from the primary structure,higher structure,physicochemical properties and bioinformatics prediction.The main findings are as follows:?1?The determination of yRBP was isolated and purified by the combination of ammonium sulfate salting-out and ion exchange chromatography.The effect of ammonium sulfate saturation on the crude extraction of riboflavin-binding protein was studied.The conditions of the ion exchange chromatography in the purification process were determined as follows:the pH value of Na₂HPO₄-NaH₂PO₄ buffer was 6.5.The concentration was 0.1mol/L.The concentration of nacl was 0.25 mol/L.The sample volume is 200 ml.The samples purified by DEAE-Toyopearl ion-exchange chromatography were identified by HPLC.The purity of the sample was 94.57%and the total recovery was 54.25%.?2?The secondary structural differences between wRBP and yRBP were determined bycircular dichroism spectroscopy,infrared spectroscopy and Raman spectroscopy.It was found that the secondary structures of wRBP and yRBP are made up of?-helix mainly,followed by random curl,?-sheet and?-turn.The wRBP contained 30.4%alpha-helix,31.8%beta-sheet,9.3%beta-turn,and 28.5%random coil.The yRBP contained 24.5%alpha-helix,39.0%beta-sheet,8.2%beta-turn,and 28.3%random coil.Although the secondary structure content obtained by different methods is not quite the same,the overall trend shows little difference in the secondary structure of wRBP and yRBP.?3?In this experiment,the amino acids of wRBP and yRBP were determined,the tertiary structure of the wRBP and yRBP was modeled by homology modeling to study the differences of wRBP and yRBP on high-level structure.4LRH was selected as the template for the construction of wRBP and yRBP.The initial structure was optimized by Discovery Studio 4.5.Finally,the model was evaluated by SAVE DOE server and then submitted to Chiron server for optimization.Based on the results,obtained the structure of model to get higher credibility.The results show that the lack of 13 amino acid residues in the yRBP compared with wRBP,due to different post-translational modifications.Through the homology modeling comparative to analysis the high structure of wRBP and yRBP,we found that there are 5?-helical structure and 2?-sheet structure in wRBP,while 6?-helical structure and 4?-sheet structure in yRBP.These structural differences have no effect on the binding site of riboflavin-binding protein to riboflavin,but affect the microenvironment in which the two proteins are located.?4?This experiment mainly studied the differences of physicochemical properties of wRBP and yRBP.The results showed that the absolute value of Zeta potential of wRBP and yRBP was 45.21 and 47.36.At pH 4,the Zeta potential of wRBP and yRBP reached an absolute value of nearly 50,indicating that the wRBP and yRBP are in a stable state.The electrical point of wRBP and yRBP is around pH 4.According to the relationship between peak time and peak area and hydrophobicity of wRBP and yRBP by HPLC,the peak area ratio of wRBP and yRBP was 0.40713 and 0.41257 respectively.This indicates that wRBP and yRBP have a lower hydrophobicity and their hydrophilicity is basically the same.The differential scanning calorimetry was used to analyze the thermal stability of wRBP and yRBP.The results showed that the denaturation temperature of wRBP and yRBP was about 68.32?,indicating that wRBP and yRBP have a high thermal stability.The fluorescence quenching mechanism,binding constants,binding sites and force types of wRBP and yRBP were studied.It was found that the optical quenching of wRBP and yRBP with riboflavin is in line with the static quenching mechanism.The binding constant Ka were 5.61 and 5.35 respectively.There is a binding sites.And the major forces between wRBP and yRBP between riboflavin are hydrogen bonds and van der Waals forces.?5?In this study,bioinformatics methods were used to predict and differentiate of wRBP and yRBP.The study found that the predicted results of wRBP and yRBP are same.wRBP and yRBP is a hydrophilic protein,is a member of the folic acid receptor family,which does not exist signal peptide.There is no transmembrane region in wRBP and yRBP.They are an outside protein.wRBP and yRBP,which are made up of?-helix,has three identical N-glycosylation sites and eight phosphorylation sites.Meanwhile,wRBP and yRBP can interact with ENSGALG00000012085,ACTR10,LMAN1,IMPDH2,FGF3,SLC19A2,SLC19A1,ENSGALG00000003025,SLC19A3.wRBP and yRBP can bind to actin and transfers the desired riboflavin to the oocyte during embryonic development.They are an essential protein in embryonic development.