Study On Isolation And Purification Of Chicken Egg White Riboflavin Binding Protein And Interaction With Riboflavin And Cu(Ⅱ)

Riboflavin binding protein(RBP) is a unique glycophosphoprotein present in eggs. It has one riboflavin and Cu(II) binding sites, respectively, play an important role during embryonic development. But the mechanism of interaction between RBP and riboflavin/Cu(II) was still not perfect. So in this paper, the riboflavin binding protein was extacted by (NH4)2SO4salting out and ion exchange chromatography. Then study the interaction between RBP and riboflavin/Cu(II) using fluorescence spectroscopy, UV-visible spectroscopy, fourier transform infrared spectroscopy. The main investigated results are listed as followed:1. The egg white riboflavin binding protein was fractionated by (NH4)2SO4salting out and ion exchange chromatography. The effects of (NH44)2SO4saturation on the crude extracts of riboflavin binding protein was studied, and the results showed that,55%-85%two step (NH4)2SO4salting-out can remove part of ovoalbumin, ovotransferrin and almost all of the ovomucin. The optimum condition of ion-exchange chromatography were:0.1mol/L Na2HPO4-NaH2PO4buffer pH6.5, NaCl elution concentration was0.25mol/L, sample volume was200mL. The purity and total recovery of riboflavin binding protein isolated by ammonium sulfate precipitation with ion-exchange chromatography were95.32%and57.36%, respectively. The activity of riboflavin binding protein extracted by this methods investigated by fluorescence spectroscopy, the results showed that protein retained the activity of combination with riboflavin.2. To elucidate the mechanism of the combination of Apo-RBP and riboflavin, the interaction between riboflavin and egg white Apo-RBP was investigated using fluorescence spectroscopy, UV-visible spectroscopy, fourier transform infrared spectroscopy. The fluorescence spectroscopy experimental results showed that riboflavin has the ability to quench the intrinsic fluorescence of Apo-RBP because of a complex formed, and the quenching mechanism is static quenching, the binding constants were5.35×108L-mol-1. Both of the thermodynamic parameters AH(-88.87kJ/mol) and AS(-131.34J/(K·mol)) were less than zero, which suggested hydrogen bonds and van der Waals played a major role in the interaction. Meanwhile, the distance and efficiency of energy transfer between riboflavin and RBP were0.70nm and0.39, respectively, based on the theory of Forster nonradiative energy transfer. The results of UV-visible spectroscopy and synchronous fluorescence spectroscopy showed that the microenvironment of Apo-RBP aromatic amino acid residues enhanced hydrophobic after interaction with riboflavin. The secondary structure of Apo-RBP and Holo-RBP were further investigated by FI-IR spectroscopy, the results showed that, binding with riboflavin, the secondary structure of RBP did not change significantly (P>0.05). Finally investigated the effects of metal ion on the binding constants between Apo-RBP and riboflavin, results showed that the binding constants of riboflavin and Apo-RBP change to3.26×109L-mol-1,3.69×108L-mol-1,0.72×107L-mol-1and9.83×105L-mol-1, respectively after adding Zn2+, Cu2+, K+, Ca2+, this indicated that Zn2+can enhanced the binding constants, Ca2+、K+will reduced the binding constants, Cu2+had little effect.3.The interaction between Cu(II) and Apo-RBP was investigated using spectroscopy, the experimental results showed that the fluorescence quenching mechanism between Cu(II) and Apo-RBP was static quenching. Investigated the effect of temperature, pH, riboflavin, concentration of NaCl on the inding constants between Apo-RBP and Cu(II). The results showed that pH had great influence on the binding constants, with the increase of pH values the association constants increased firstly and then decreased gradually. The optimum pH for Cu(II) binding to Apo-RBP were at pH7.0-8.0; temperature and NaCl concentration did not has significantly effects; the binding site of riboflavin in Apo-RBP is not the same with Cu(II) we spectulated by the effect of riboflavin saturation of RBP on the interaction between Cu(II) and Apo-RBP. In addition, the ANS experiments showed that the binding sites of Cu(II) may be located in the hydrophobic region of Apo-RBP. The effect of Cu(II) on the conformational changes of Apo-RBP was investigated with UV-visible absorption spectroscopy, synchronous fluorescence spectroscopy and fourier transform infrared spectroscopy. The results showed tha the hydrophobic of Apo-RBP tyrosine residues micro-environment was increased, the a-helix,β-sheet and β-turn decreased from26.45%to17.48%,18.15%to14.01%, and18.40%to14.01%, respectively. Meanwhile, the random coil increased from13.05%to23.91%, its explained the structure of Apo-RBP transition from order to disorder after interaction with Cu(II).4.To further investigate the binding mechanism of Cu(II) and Apo-RBP, the interaction between Cu(II) and Apo-RBP was investigated using cyclic voltammetry and differential pulse voltammetry. The results showed that there were two oxidation peaks and two reduction peaks of Cu(II) in pH7.0Tris-HCl buffer, but Apo-RBP had no oxidation or reduction peaks in the scan range of0.2V to-1.0V, indicating that Apo-RBP had non-electrochemical activity on glassy carbon electrode surface. With the increase of the concentration of Apo-RBP, the two oxidation peaks current decreased from11.23μA and6.19μA to5.63μA and3.24μA, respectively, and the two reduction peaks current decreased from5.03μA and14.28μA to3.03μA and11.02μA, respectively, meanwhile, two oxidation peaks current of differential pulse voltammetric was gradually decreased from16.14μA and15.69μA to15.69μA and5.55μA, respectively. The results indicated that Cu(II) and Apo-RBP was formation of a non-electrochemical activity compound so the Apo-RBP can stable the redox state of Cu(II), to prevent harmful oxidation-reduction reaction during storage of eggs.