摘要
我們設計不具二級結構之短鏈胜肽及選用不同疏水性之吸附劑，探討生化分子間之疏水性交互作用。此一研究利用疏水性層析之操作方式，探討胜肽鏈中tryptophan含量對疏水性之影響、各種胺基酸（烷基側鏈胺基酸及帶電荷胺基酸）對疏水性吸附之影響、胜肽鏈中疏水胺基酸序列對疏水性之影響、雙官能基吸附劑上烷基鏈長及烷基鏈密度對疏水性交互作用之影響。 我們將各種胺基酸X插入GWWG胜肽鏈中之兩tryptophan間，以層析方式分析各種胺基酸在胜肽鏈中之疏水表現。實驗結果顯示，tryptophan與吸附劑上之octyl疏水基具有相當強的疏水性交互作用，胜肽鏈中每一tryptophan所貢獻之放熱量約9.4～11.8 kJ/mole。相反的，烷基側鏈胺基酸之疏水性貢獻，主要來自去水合程序所引起之亂度上升。 將帶負電荷胺基酸插入GWWG中，使得胜肽與octyl烷基鏈間之疏水作用力降低。我們認為係因圍繞於帶負電荷胺基酸側鏈之水分子排列並不緊密，故移除水分子時亂度並未大幅提高，以致於滯留因子低於GWWG。 插入帶正電荷胺基酸lysine於GWWG中，雖然與吸附劑上之octyl烷基鏈有交互作用，但卻降低了tryptophan與octyl烷基鏈間之直接作用，又tryptophan與octyl烷基鏈間因凡得瓦爾作用所釋放之熱能較lysine胺基酸多，而降低tryptophan與octyl烷基鏈間之交互作用，吸附焓將因而上升，同時也導致自由能上升，是故滯留因子降低。 我們也探討疏水胺基酸排序對疏水性之影響。實驗結果顯示，tryptophan位於胜肽鏈中間位置疏水性最高，位於羧基端次之，tryptophan位於胺基端其疏水性最低。 據由吸附劑上烷基鏈長及鏈密度對疏水作用影響之探討，我們得知隨烷基鏈長或密度增加，滯留因子亦隨之增加。然而，吸附焓隨吸附劑上烷基鏈長增加而呈指數下降。當烷基鏈長由己基(Hexyl)增長為辛基(Octyl)，胜肽之滯留性大幅提高，但烷基鏈長超過辛基後，其增加趨緩。我們認為，滯留性趨緩乃受吸附劑上所含之烷基鏈之構形改變，所引發之亂度變化影響所致。我們也探討基材上所含之羧基對於疏水性交互作用的影響。結果顯示，當基材上含負電荷時，即使操作在高鹽濃度下也會影響胜肽析出次序。
並列摘要
Five amino acid peptides were used to study the hydrophobic contribution of each different amino acid in the peptide. The peptides had two glycines on both the N and C terminal of the peptides. Two tryptophans were right next to the end capped glycines. Each different amino acid was inserted between the two tryptophans. Chromatographic method was adopted to evaluate the contribution of each amino acid on peptide’s retention, adsorption heat, adsorption entropy and the number of expelled water after adsorption. The capacity factors of peptides on an Octyl-Sepharose column were estimated. The adsorption heat was evaluated by linear van Hoff’ plot and the number of expelled water was estimated by a simplified preferential interaction model. The number of expelled water provided valuable information of the relative size of contacting area between peptides and the octyl ligands on adsorbents. The adsorption enthalpy and entropy helped us to understand the contribution of water expelling on hydrophobic adsorption and how each amino acid affected the adsorption. Firstly, we study the hydrophobic contribution of tryptophan. It was found that tryptophan interacted strongly with octyl ligands and each tryptophan in peptide released 9.4-11.8 kJ/mole heat upon adsorption. On the contrary, the aliphatic amino acids, although facilitated hydrophobic adsorption, absorbed heat upon adsorption. It was clearly that the contribution of aliphatic amino acids on hydrophobic adsorption was through the increase in system entropy by expelling more ordered water. The insertion of negatively charged amino acids in the middle of GWWG resulted in the reduction of capacity factor. The number of expelled water showed there was sufficient direct contact between amino acids and octyl ligands. It was suspected that the water surrounding the side chains of ionic amino acids was not orderly aligned. Therefore, the expelling of water did not provide enough entropy elevation to enhance adsorption. The insertion of positively charged lysine also inhibited hydrophobic interaction. The number of expelled water was even less than the peptide GWGWG. It indicated that lysine tended to reduce the interactions between tryptophan and ligands. Since the interaction between tryptophan and ligands was highly exothermic, the reduction of tryptophan-ligand interaction would lead to an increase in adsorption enthalpy. The increase in adsorption enthalpy, in turn, would lead to an increase in Gibbs free energy, and ultimately the decrease of capacity factor. We also studied the effect of amino acid sequence on the hydrophobicity of short chain peptides. The results showed that tryptophan, the most hydrophobic amino acid, when located in the middle of the peptide chain, made the peptide more hydrophobic than when it was located at the ends of the peptide, especially at the amino end. Hydrophobic interactions between biomolecules and ligands were also influenced by ligand chain length and ligand density. The capacity factor increased as the alkyl chain length and density increased. The capacity factor seems to increase linearly with the increasing of ligand density; meanwhile, the adsorption enthalpy and entropy decrease linearly. However, the adsorption enthalpy decreased exponentially with the ligand length and there was a sudden increase in capacity factor when the alkyl chain length increased from 6-carbon to 8-carbon. The results showed that the entropy alkyl chain was also affected the adsorption. The effect of residual charges on the adsorption matrix was also studied. It was also found that the hydrophobic interactions between peptides and Alkyl-CM-Sepharose were affected by the presence of the negatively charged carboxymethyl groups on the resin. Even at high salt concentrations, the elution order of peptides was affected by these negative charges on the resin.