生物膜主要由脂質(lipid)構成，在本實驗中將以POPC/POPG(3/1)混合的帶負電荷脂質微胞--SUV(small unilamellar vesicle，尺寸約為30nm)，作為模擬生物細胞膜的簡單模型，研究帶正電的抗菌小蛋白(peptide)其間的交互作用與濃度的關係。 本實驗設計將SUV溶液逐次滴入Ma2-amide溶液中，量測在30°C、pH=7.4的環境下兩者的吸附。利用circular dichroism (CD)量測peptide在不同吸附狀態的特徵光譜，分析出參與吸附的peptide數量，與吸附所造成的自由能變化—△G；另利用isothermal titration calorimetry (ITC)量測吸附所造成的熱量變化—△H。 依據多體效應的理論模型，吸附放熱隨Pb/L的變化將具1)膜變薄效應與2)膜電荷中合效應，此兩項皆會使放熱減小。代入數據分析，發現以此L滴定P的實驗方法，得到的放熱變化值遠大於理論值，且綜合Pb的變化後發現，當Pb不再增加時仍有大量放熱反應，因此推斷本實驗中有除吸附造成的多體效應外，另有Pb轉移不同脂質微胞膜面的反應。 The main components of cell membranes is lipid.In this investigation, we use the lipide vesicles --SUV (small unilamellar vesicle, with size around 30nm) with POPC/POPG(3/1) mixture, that have negative surface charge as our membrane models to study the thermaldynamics of antimicrobial peptide(called peptide, that have positive charge) binding to membranes. In this investigation, the solution of antimicrobial peptide (Ma2-amide, purified from frog) were titrated by a series of SUV solution, to study the binding of lipid and peptide at 30°C and pH=7.4 environment. Circular dichroism (CD) can measure the spectrum of peptide binding in membrane and free in buffer, from this binding isotherm to analyze the free energy change △G from binding. In isothermal titration calorimetry (ITC), the binding enthalpy ΔH will measure, and according to binding isotherm, we can get the binding enthalpy per binding peptide. Peptides binding to membrane have two types of many-body effect, (1) membrane thinning effect, and (2) membrane surface charge neutralized effect. Both of two will reduce ΔH (being less negative) as bound peptides increase. But in this L into P experiment, ΔH decrease much faster then the many-body effect. There are other exothermic reactions occur – binding peptide transfer to new SUV in L into P experiment.
