- 學位論文
雙功能吸附基材在蛋白質置換層析的應用
Application of bifunctional adsorbent in protein displacement chromatography
摘要
置換層析是一種既經濟又快速的生化分離技術，不但有高產率且能同時分離和濃縮生化分子。近十年來，許多研究已成功的應用於置換層析分離蛋白質，但是常由於缺乏適當的置換劑，且蛋白質不易與置換劑分離，加上管柱再生不易，使得置換層析無法廣泛的被應用。 本研究主要是運用疏水性作用力來操作蛋白質置換層析，其中我們探討了核糖核酸脢(Ribonuclease A)、胰蛋白分解酵素原(Trypsinogen)、牛血清蛋白(BSA)與血紅素(Hemoglobin)對吸附基材的恆溫吸附曲線，可了解各蛋白質在基材上之最大吸附量與親和力之關係。結果發現Hemoglobin與Trypsinogen的吸附量較大，且隨著Ligand密度增加時其最大吸附量僅微幅上升，而BSA與RNase A則隨Ligand密度增加其最大吸附量大幅上升。在與基材之親和力方面，隨著Ligand密度增加時，Hemoglobin與BSA的親和力略微增加，Trypsinogen則是大幅增加，而RNase A則是減少。因此我們推測Trypsinogen 與Hemoglobin比BSA與RNase A有較大的疏水表面積。 由恆溫吸附曲線中可發現Trypsinogen與BSA在濃度為0.12mM時有交錯的現象。以BSA為置換劑時，可發現置換劑濃度低於0.12mM時其分離效果較好；置換劑濃度高於0.12mM時，無法置換Trypsinogen，因此分離效果較差。 我們選用分子量較大且價格較廉的牛血清蛋白(BSA)與血紅素(Hemoglobin)為置換劑，嚐試置換分離分子量較小的胰蛋白分解酵素原(Trypsinogen)與核糖核酸脢(Ribonuclease A)二種蛋白質之混合物。其中可發現分子量大小之差異也會影響置換的行為，以Hemoglobin為置換劑我們可發現其吸附量與親和力均比Trypsinogen大，但卻無法完全將Trypsinogen置換，因此我們猜測是分子量大小差異太大的原因。為了證實此現象，我們以Hemoglobin為置換劑置換與其分子量相似的BSA，確實發現有較好的置換效果 另外，為了提升管柱再生的效率，我們合成了含有陰離子之疏水性雙功能吸附基材(Butyl-CM-Sepharose，61μmole/mL gel)進行管柱再生，由再生實驗的結果我們可得知置換劑是BSA (牛血清蛋白)之再生率為90.80%而Hemoglobin (血紅素)之再生率為86.55%，因此我們可以確定以提升pH值的方式在管柱再生方面也有不錯的效果。
並列摘要
Many studies have underscored the potential of displacement chromatography as an economic high-throughput preparative technique and the ability to carry out simultaneous concentration and purification. However, separation become difficult when the adsorption isotherms of two species cross. To investigate the separation performance when there exists isotherm cross, the adsorption isotherms of proteins, ribonuclease A (RNase A), trypsinogen, bovine serum albumin (BSA) and hemoglobin, on butyl-containing hydrophobic adsorbents were measured. It was found that the maximum capacity of hemoglobin and trypsinogen increase only slightly as the butyl density increased. But the affinity of trypsinogen increased drastically with the increase in ligand density. On the contrary the maximum capacity of BSA and RNase A greatly increased with the ligand density. But the affinity of RNase A decreased slightly with the increase in ligand density. It indicates that trypsinogen and hemoglobin own larger but BSA and RNase A own more specific hydrophobic surface area. The adsorption isotherms of trypsinogen and BSA crossed at a protein concentration of 0.12 mM when they were adsorbed on an adsorbent containing 61 mM of butyl groups. When BSA was used as the displacer, it was found that the best displacement occurred when the concentration of BSA was lower than 0.12 mM. A displacer concentration higher than 0.12 mM would not be able to displace trypsinogen. It was found that the size difference between displacer and adsorbate also affected the displacement performance. When we used hemoglobin as the displacer, of which the maximum capacity and affinity were higher than those of trypsinogen, we still cannot obtain satisfactory displacement. We suspect that it was due to the much larger molecular size of BSA and hemoglobin that trypsinogen can still adsorb on the space between displacers. To demonstrate this point we tried to displace BSA by hemoglobin since they have similar molecular mass. It was found that hemoglobin could fully displace BSA. After the displacement operation,the adsorbents which were saturated with displacers need to be regenerated. The displacer, BSA or hemoglobin, could be easily recovered from the Butyl-CM-Sepharose (61μmole/ml gel) by slightly elevated pH. The recovery of hemoglobin and BSA were 86.55% and 90.80%, respectively.
參考文獻
延伸閱讀
- 柯玲潔（2006）。以雙功能吸附基材進行蛋白質復性—疏水基鏈長與密度之選擇〔碩士論文，中原大學〕。華藝線上圖書館。https://doi.org/10.6840/cycu200600172
- 黃媺晴（2015）。探討生物界面之蛋白質吸附機制及其在建構功能性生醫材料之應用〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2015.10617
- 鍾武傑（2007）。利用雙功能吸附基材進行蛋白復性-蛋白吸附狀態對復性的影響〔碩士論文，國立中央大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0031-0207200917350848
- Xu, A., Wang, X., Fa, Y., Liu, Y., Li, F., Huang, H., & Wang, Z. (2017). Determination of Bicarbonate in Fermentation of Cyanobacterial by Non-Suppress Ion Chromatography. Advance Journal of Food Science and Technology, 13(1), 7-10. https://doi.org/10.19026/ajfst.13.3410
- 張雅茹（2012）。Application of Bioactive Mesoporosus Silica on the Detection of Antibody on the Lateral-Flow Immunochromatography〔碩士論文，中原大學〕。華藝線上圖書館。https://doi.org/10.6840/cycu201200596