Measurement Of Protein Surface Hydrophobicity By Fluorescence Probe Method And Hydrophobic Interaction Chromatography

Surface hydrophobicity is an important property of proteins and plays a significant role in maintaining the conformation and ensuring functional activity for proteins. It is also a theoretical basis of hydrophobic interaction chromatography (HIC) during the purification of recombinant proteins, antibodies and other proteins. Knowledge about the effects of various factors on the surface hydrophobicity of proteins would be helpful to understand the changes in physiological activity and conformation of proteins and also useful to optimize the operation conditions for protein separation and purification process. Therefore, in the present work, the variation of proteins’surface hydrophobicities under different conditions has been studied with two methods, fluorescence probe and HIC. Main results are listed as follows.Firstly,8-anilino-l-naphthalene sulfonate ammonium salt (ANS) was used as the fluorescence probe to measure the surface hydrophobicity indexes (So) of bovine serum albumin (BSA) and bovine immunoglobulin (bIgG) in different solutions. The appropriate pH was chosen as7.4. The influences of temperature, salts, denaturants and sodium caprylate (NaCA) on the proteins’surface hydrophobicities were investigated. Heating showed opposite effects on the surface hydrophobicities of BSA and bIgG. The addition of kosmotropic salts (Na2SO4and (NH4)2SO4), and neutral salt (NaCl) would cause the increase of So value for both BSA and bIgG, while chaotropic salt (NaSCN) dramatically reduced So values. There was a big difference between the effects of sodium dodecyl sulfonate (SDS) on the surface hydrophobicities of two proteins. Guanidine hydrochloride (GdnHCl) caused a decline in So value for BSA, while So value of bIgG increased in low GdnHCl concentration and decreased as concentration increased. The binding of NaCA with hydrophobic pockets on BSA led to decrease of S0value for BSA.Secondly, the interaction between ANS and BSA or bIgG was analyzed by fluorescence spectrometry. Several data treatment methods were investigated. The binding sites and binding constants of BSA-ANS and bIgG-ANS system were obtained through the analysis based on Scatchard’s equation, which were5.8and10.7,3.37x106M-1and3.34x104M-1respectively. In addition, Adair’s equation was also used to analyze the BSA-ANS system which could provide binding sites and binding constants for each site.Finally, HIC was applied to determine capacity factors (k’) of BSA, bIgG and lysozyme on Phenyl Sepharose6Fast Flow column in different mobile phases. The impacts of salts were discussed by the preferential interaction theory. In the presence of kosmotropic salt like (NH4)2SO4and neutral salt such as NaCl, total numbers of water molecules released (-△v1) during the binding process were positive for three proteins tested. In contrast to the results obtained with (NH4)2SO4and NaCl,-△v1for three proteins were negative when chaotropic salt (NaSCN) existed. For lysozyme and BSA, when pH of mobile phase was closer to their pIs, they showed high κ’While the effects of pH on bIgG showed some significant difference. The k’ values declined as the concentration of guanidine hydrochloride increased. With NaCA concentration varying from0to75mM, k’value of BSA showed a decline first and then increased, while that of bIgG almost remained unchanged. As the amount of alcohol increased, capacity factors of three proteins decreased.Two methods, fluorescence probe and HIC, were compared to determine the protein surface hydrophobicity. Good relationships could be found between So and k’under some conditions. Same conclusion could be drawn by the two methods upon the influences of different salts. So value of BSA decreased with increasing GdnHCl concentration which was consistent with the results obtained from HIC experiments. With the same concentration ratio of NaCA to BSA, the variation of So value for BSA was the same with the change of κ’with NaCA concentration ranging from0to50mM. The results indicated that the surface hydrophobicity index of proteins determined by fluorescence probe method could be used to predict the retention behavior of proteins in HIC column with corresponding mobile phase.Generally, in this paper, the variation of surface hydrophobicities of BSA and bIgG in different solutions were investigated by fluorescence probe and HIC, and the changes in surface hydrophobicity of lysozyme by HIC, and some rules have been obtained. On one hand, the changes of protein surface hydrophobicity can provide some information about the changes in the protein structure under different conditions which could help to provide guidance for the storage of protein solution. On the other hand, the protein surface hydrophobicity can be used to predict retention behavior of proteins in HIC and hydrophobic charge-induction chromatography (HCIC) and then improve the separation efficiency.