探討精胺酸對蛋白質聚集之抑制作用

Abstract

精胺酸(L-arginine)屬於半必需(semi essential)胺基酸，是種生物相容性(biologically compatible)分子，不會對人體產生傷害。許多研究指出精胺酸可有效減少蛋白質聚集體之產生，不過其確切之聚集抑制機制仍未被清楚地了解。 本論文之第一部份以木瓜酵素(papain)為 model protein，探討精胺酸對不規則聚集體形成(disordered aggregates)之抑制作用。研究結果顯示，低濃度胍鹽酸會誘導酸性條件下(pH 2.0)之木瓜酵素產生聚集，且於胍鹽酸濃度0.7 M 時聚集程度最大，因此以胍鹽酸0.7 M 作為木瓜酵素酸性條件下之聚集條件。當添加入少量精胺酸(0.02-0.2 M)時，胍鹽酸誘導之木瓜酵素聚集不減反增；但當加入精胺酸濃度增加(0.3-1.5 M)後，木瓜酵素聚集量則隨之減少。因此推測精胺酸此特殊現象與胍鹽酸相似，不但具有開展木瓜酵素結構特質，且其與木瓜酵素間之作用與疏水性作用力相關。 此外，為了探討精胺酸抑制聚集時對酵素活性之回復能力，本研究更進一步 將溶液環境調整為中性(pH 7.4)，並以65°C 為聚集形成條件。研究結果發現，木瓜酵素溶液含有1M 以上精胺酸可有效地減少聚集之產生，但卻只能保存少量酵 素活性(約10 %)。推測此現象乃因木瓜酵素之active sites位於conformational flexibility較高之區域，因此木瓜酵素之活性變化比結構變化快很多，而精胺酸主要幫助減少木瓜酵素分子間聚合，故無法保存大量的酵素活性。 本論文之第二部份以牛血清蛋白(BSA)為 model protein，探討精胺酸對規則聚集體(ordered aggregates)形成之抑制作用。牛血清蛋白加熱於65°C 時β- sheet 含量增加且產生不成熟 fibril，而當使用ThT螢光強度觀察精胺酸對纖維含量之影響時發現，溶液中所含精胺酸濃度越高(0- 1.4M)，ThT螢光強度會逐漸減少。但由穿透式電子顯微鏡(TEM)、動態雷射光散射法(DLS)及大小排阻層析法(SEC)之結果發現，含有1M 精胺酸與不含精胺酸之牛血清蛋白樣本所包含之蛋白質纖維大小與含量幾乎相同，且由螢光滴定(fluorescence titration)實驗發現，精胺酸與ThT之間對牛血清蛋白纖維有競爭性鍵結(competitive binding)現象。我們研究結果除了顯示精胺酸無法抑制牛血清蛋白之纖維生成外，並指出當使用ThT螢光法研究類澱粉纖維含量時，應特別小心添加劑影響所造成之誤判情形。

L -arginine is a type of semi essential amino acid, and does not cause any harm on human being due to its biologically compatible property. While several lines of evidence has indicated that L arginine can effectively reduce protein aggregation, the precise interacting mechanism regarding the aggregation inhibition induced by L- arginine still remains elusive. The first part of this thesis is aimed at exploring the effects of L- arginine on the formation of amorphous aggregates using papain as the model protein system. Our data revealed that low concentration of guanidine hydrochloride (GdnHCl) could induce papain aggregation at acid condition (pH 2.0) with the highest level of aggregation observed at 0.7 M GdnHCl, which was taken as the aggregation condition used in our study. We found that, when a small amount of L- arginine (final concentration 0.02- 0.2M) were present in papain solution, the amount of papain aggregation was augmented. However L- arginine was observed to dose dependently attenuate the extent of papain aggregation upon futher increase of L- arginine (final concentration 0.3- 1.5M). Therefore, we postulate that, similar to GdnHCl, L- arginine not only perturbs the structure of papain but also has hydrophobic interactions with papain. In addition, in order to explore the ability of L- arginine to retain papain activity when its anti aggregation potency was observed, neutral pH and 65°C were used as the aggregation inductive condition. Our results demonstrated that, whereas 1M L-arginine was sufficient to decrease papain aggregation, only 10% of papain activity was retained. We hypothesize that papain active sites may exhibit higher conformational flexibility than the enzyme molecule as a whole, resulting in the finding that the loss of papain activity precedes noticeable conformational change of papain. As for the study of protein ordered aggregation, we used bovine serum albumin (BSA) as model protein. When incubating BSA at 65°C for hours, there was a considerable reduction in the α helical content accompanied by increases in β- sheet content in the BSA solution containing immature fibrils. We found that L- arginine dose dependently reduced the thioflavin T (ThT) fluorescence of BSA. However, as revealed by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and dynamic light scattering (DLS) results, L-arginin did not prevent amyloid- like fibril formation by BSA. We concluded that L -arginine competed against ThT for binding sites on BSA amyloid -like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.