The microcalorimetric study of the contributions of hydrophobic interaction effect: the protein binding mechanisms between proteins and resins, and the protein solution behaviors

• Main Authors: Hsiang-Ming Huang, 黃祥鳴
• Other Authors: Wen-Yih Chen
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/30678458020138688970

博士 === 國立中央大學 === 化學工程與材料工程研究所 === 91 === Abstract To understand the roles of hydrophobic interaction in the biorecognition system, we studied the simple model systems of hydrophobic interaction between protein and interaction surface in this investigation. We discussed the effects of environment factors such as temperature, hydrophobic chain length and the contribution of solution molecules in the hydrophobic interaction between biomolecules. By the thermodynamic analysis, we estabished an interaction mechanism to explain the protein-resin and protein-protein interaction behavior in solution. As summary, we divide into five related topics as following: (1)In the study of hydrophobic interaction between protein and matrix, we studied two kinds of proteins with similar molecule weight and at nearly isoelectrical point (α-Chymotrypsinogen A and Trypsinogen) of solution. Results showed that the increasing temperature significantly influence the binding mechanism of α-Chymotrypsinogen A with Butyl-Sepharose from an adsorption-dominated binding process to a partition-dominated binding process because α-Chymotrypsinogen A is a temperature sensitive protein. Evidences showed in the dilution enthalpy measurements confirmed this suggestion. (2)To understand the contribution of temperature effect on protein binding process, we chose proteins (Lysozyme、α-amylase、Myoglobin) and hydrophobic resins (Butyl-Sepharose、Octyl-Sepharose) to study the protein binding mechanism. We discussed the effects of salt, ligand chain length, protein characteristics, temperature and so on. Results showed that the increased salt concentration, hydrophobic ligand chain length and temperature promote the hydrophobic interaction between proteins and resins because the increased hydrophobic interaction regions between these suspended reactants. (3)We selected proteins (Lysozyme、Myoglobin) and cation resins (CM-Sepharose、SP-Sepharose、Source-30S) to study the contributons of hydrophobic interaction between ligand and protein in IEC system. We studied the effect of pH, salt, ligand and matrix, protein characteristics by ITC and isotherm experiments. We also utilized scatchard plot and Freundlich-Langmuir model to analyze the protein-binding model. In higher pH solution condition, results showed that the hydrophobic interaction between proteins and resins could significantly influence the protein binding process and the protein binding affinity was promoted by salt effect. In contrast, the electrostatic interaction dominated the protein binding process in lower pH solution condition and the increased salt concentration decreased the protein binding affinity. Moreover, we revealed that Source-30S, which has the most hydrophilic ligand and the most hydrophobic aromatic matrix, could make stronger hydrophobic interaction with protein in different solution conditions. (4)To study the effect of aromatic ligand chain length in the peptide-resin binding process, we designed three kinds of peptides with different tryptophan chain length (Gly-Trp-Gly：GWG、Gly-Trp-Trp-Gly：GWWG、Gly-Trp-Trp-Trp-Gly：GWWG). Results showed that GWG has stronger binding affinity with hydrophobic resins, and the dilution of GWG is an endothermic reaction. In contrast, the dilutions of GWWG or GWWWG are exothermic reactions. This is because the increased tryptophan on the peptides increase the cation-π between peptide and solution molecules. Furthermore, we demonstrated that the hydrophobic interaction between GWWWG and CM-Sepharose dominated the peptide binding process whereas GWG and GWWG interact with CM-Sepharose is a electrostatic dominated binding process. (5)We also discussed the protein solution dilution behaviors in varied lysozyme concentration. At higher protein concentration (25g/L), we found that the stronger protein-protein interactions make an energy unfavorite protein dilution reaction. In contrast, the dilution of lysozyme at lower protein concentration (5g/L) is an exothermic reaction because the existence of electrostatic repulsion between protein molecules. Furthermore, we observed the decreased electrostatic repulsion between protein molecules at higher salt concentration could result in the increased protein dilution enthalpy. In this investigation, we studied the protein solution behavior and protein binding mechanism in varied solution conditions by thermodynamic analysis. The results and discussions provide useful knowledge to understand the protein binding behaviors in solution and fundamental thermodynamic information in biorecognition system.