利用原子力顯微術偵測單一生物分子間專一性之鍵結強度、解離及其熱力學之研究

Specific Recognition Force, Dissociation and Thermodynamics of Single-pair Antibody-Antigen Interaction Using Atomic Force Microscopy

王裕銘(Yu-Ming Wang)

指導教授 ： 黃榮山 ； 林世明

國立臺灣大學/工學院/應用力學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.00554

摘要

生物分子藉由彼此間的辨識及鍵結過程在生命體中進行訊息傳遞、酵素催化及免疫保護等生化反應過程。為探討生物分子間的交互作用，本論文利用原子力顯微術進行免疫球蛋白及其抗體與升糖素及其抗體在不同酸鹼值、環境溫度及解離速度等條件下量測其解離力、分子間解離動力常數及熱力學參數。此結果可提供直接的證據去解釋分子間在更接近事實且不同環境下的行為。
本研究將免疫球蛋白及其抗體分別共價鍵結於原子力顯微鏡之探針與玻片並利用力曲線進行解離力在不同酸鹼值溶液下檢測，其結果顯示解離力在中性溶液擁有最佳鍵結強度。隨著增加或降低溶液之pH值皆會使生物分子間交互作用力下降。該結果可歸因於生物分子之構形在不同酸鹼值溶液下會產生不同形變，而造成分子間不完全的接合。此外，生物分子之官能基與生物功能皆會隨著溶液酸鹼值改變而改變，進而影響其抗體之辨識。而在分析的解離速率與自由能也證實了生物分子在中性溶液具有低解離速率及高能障等特點，使生物分子間能穩定的鍵結相較於在酸性或鹼性溶液下。在環境溫度變化對免疫球蛋白間鍵結的實驗結果中顯示分子間之交互作用力會隨著度增加而降低，此可歸因於溫度變化會引起分子構形改變造成生物分子間無法緊密皆合及溫度所造成的布朗運動碰撞分子間的鍵結，使鍵結強度降低。藉由改變環境溫度可檢測生物分子之解離熵及解離焓，其結果顯示解離熵及解離焓均會隨著溫度上升而增加，此說明了溫度會造成分子結構鬆散進而影響分子間的交互作用。
在升糖素及其抗體的研究中也顯示了生物分子間鍵結強度會隨著環境酸鹼值的增加或降低而下降，此結果說明了分子結構會受溶液中之離子影響進而使分子間交互作用力下降。此結果也可從所估算之自由能強度及解離速率得到印證。在溫度變化之實驗中發現分子間鍵結力會隨著溫度上升而線性下降。此可歸因於升糖素為構形簡單之胺基酸長鏈所組成，因此與溫度會產生強烈之關聯性。此外，在熱動力參數發現升糖素及其抗體在35 0C會急劇上升，此說明了分子構形及鍵結性質均在35 0C產生變化。
本論文已成功地利用原子力顯微術進行生物分子間鍵結強度、解離速率、活化能、解離熵及解離焓之量測。其結果可被應用在生物檢測晶片之生物分子解離環境及探討生命科學之依據。

關鍵字

原子力顯微鏡 ； 單一生物分子間交互作用力 ； 自由能 ； 解離熵 ； 解離焓

並列摘要

Molecular recognition and intermolecular binding are essential for implementing many biochemical and biological processes in living organisms. To further understand the molecular binding mechanisms, this study used atomic force microscopy as a force-based senor for investigation of the force strength between antibody and antigen complex in a single pair level with varied physiological (pH and temperature) and physical (loading rate) conditions. The results provided direct evidence of unbinding force, dissociation rate and thermodynamic parameters that explained intermolecular behavior of human IgG1/anti-human IgG1 complex and glucagon/anti-glucagon IgG complexes, respectively.
Mean measured forces of human IgG1 and its specific antibody system with pH-varied liquid environments showed a sharp decrease with a decrease of pH value (acidic environment), and a gradual decrease with an increase of pH value (alkaline environment) from a reference level at neutrality. This could have corresponded to the pH-induced change in conformational change and outer functional groups of amino acids which are protonated. As a result of change in pH environment of human IgG1/anti-human IgG1 complex, surface protonated properties and conformation weakened intermolecular force. Molecular dynamic behavior and free energy change were also contributed to a high probability of bonds breaking and a low magnitude of energy barrier when molecules were immersed in acidic or alkaline solution. Temperature-dependent unbinding force experiments were also carried out. The results showed that the unbinding forces decreased with an increase of environmental temperatures. This could be largely due to temperature-induced conformational change in volume expansion and strong Brownian motion. As a result, interaction forces decreased. Estimated dynamic behavior and thermodynamic parameters also showed weak interactions under high temperatures. This could have corresponded to looser molecular structure and weaker intermolecular interaction in high temperature, thereby increasing entropy and enthalpy.
The interaction between glucagon and anti-glucagon IgG with pH- varied liquid environment exhibited weak interaction force, low energy barriers and high dissociation rates under acidic and alkaline solutions. This indicated that molecular interactions turned out weak forces when pH values increased or decreased away from neutrality. Force measurement as a function of temperature exhibited a nearly linear decrease of force strength with an increase of temperature. This could have been attributed to molecular charge-free conformational changes, resulting in incomplete binding. The thermodynamic enthalpy and entropy for interaction showed an increase with increasing temperature.
Specific interactions of human IgG1/anti-human IgG1 pairs and glucagon/anti-glucagon IgG pairs have been successfully investigated by the atomic force microscope. With the use of an extended Bell and Evans model, the molecular dynamic behavior, free energy change and thermodynamic parameters can be obtained by varying physiological (pH and temperature) and physical (loading rate) conditions. The results provided directly evidence to explain the biological interactions.

並列關鍵字

atomic force microscopy ； dissociation rate ； enthalpy ； entropy

參考文獻

[1] J.W. Weisel, H. Shuman, R.I. Litvinov, Protein-protein unbinding induced by force: single-molecule studies, Current Opinion in Structural Biology 13 (2003) 227-235.

[2] D.R. Davies, E.A. Padlan, Antibody-antigen complexes, Annu. Rev. Biochem. 59 (1990) 439-473.

[3] A. Skerra, Imitating the humoral immune response, Current Opinion in Chemical Biology 7 (2003) 683-693.

[5] A.W.P. Vermeer, W. Norde, The thermal stability of Immunoglobuli: unfolding and aggregation of a multi-domain protein, Biophy. J. 78 (2000) 394-404.

[6] J. Buchner, M. Renner, H. Lilie, H.J. Hinz, R. Jaenicke, Alternatively folded states of an immunoglobulin, Biochemistry 30 (1991) 6922-6929.