飛秒雷射誘發胺基酸與蛋白質於氣液介面結晶化之研究

Abstract

使用雷射誘使結晶化的發生已成為現今取得分子結晶相當重要的技術，特別是已於2002年被成功示範的飛秒雷射誘使結晶化技術，現正往蛋白質結晶迅速發展。當高強度的雷射脈衝被聚焦於過飽和水溶液之中，水的多光子吸收現象發生，並對周圍環境產生機械性壓力波，最後致使溶質結晶。於本研究中，我們探討飛秒雷射參數如脈衝能量、脈衝頻率與聚焦位置對結晶化與取得結晶的影響，並示範利用聚焦雷射於溶液表面可有效提升分子結晶的機率。 在第一部分，我們示範飛秒雷射誘發甘胺酸過飽和溶液結晶化並檢驗雷射參數與結晶機率、結晶形貌和晶相的關係。脈衝能量與頻率的依存性實驗結果顯示結晶形貌與數量主要是由水的多光子吸收現象引發的空穴氣泡產生頻率所決定。此外，藉由聚焦雷射脈衝於空氣溶液介面可大幅增進結晶機率，其原因被推斷可能為甘胺酸分子於介面的分子吸附，並依此推論以我們所知首次成功示範使用單發雷射脈衝誘發甘胺酸單晶的產生。 於第二部分，我們以類似上述的方式示範飛秒雷射誘發於蛋白質結晶學中最標準的酵素與蛋白質-溶菌酶過飽和溶液結晶化。我們發現相較於聚焦雷射在溶液液滴中與自然結晶的條件下，聚焦雷射在溶液液滴的空氣溶液介面可顯著地增進結晶機率。另從各條件下取得之結晶的X-ray繞射分析結果可得知其晶格結構並無差異。儘管有研究指出蛋白質會吸附於溶液表面且會致使蛋白質變性，但實驗結果顯示鹽析效應可穩定蛋白質於溶液介面的結構，而相關的當代研究亦支持此結果。 目前的研究結果表示於飛秒雷射誘發結晶化中使用雷射照射溶液表面可改善現存的結晶技術。分子在溶液表面成核的初期階段的進一步光譜動態研究可望從此結晶方法建立新穎的表面/介面分子科學。

Laser-induced crystallization is now becoming an indispensable technique to obtain molecular crystal. Particularly, femtosecond (fs) laser-induced crystallization was demonstrated in 2002 and is developing especially toward protein crystallization technique. When an intense fs laser pulse is focused into a supersaturated solution, multiphoton absorption of solvent occurs and induces mechanical stress to the surrounding area, which results in crystallization of solute. In this study, we explore how fs laser parameters such as pulse energy, repetition rate, and focal position affect crystallization and obtained crystals, and then demonstrate that solution surface irradiation of fs laser pulses can improve molecular crystallization probability drastically. Firstly, fs laser induced crystallization of glycine from the supersaturated solution is demonstrated and relations among laser parameters and crystallization probability, crystal morphology, and the polymorph are examined. Pulse energy and repetition rate dependences show that the frequency of cavitation bubble generation induced by multiphoton absorption of water mainly determines crystal morphology and number. Furthermore, significant increase of crystallization probability is also demonstrated by focusing fs pulses at the air/solution interface, whose mechanism may be ascribed to molecular adsorption at the interface. Based on the inference, we have succeeded in single glycine crystal formation induced by single fs pulse irradiation for the first time as far as we know. Secondly, we demonstrate fs laser induced crystallization of lysozyme as the most standard enzyme and protein in protein crystallography in the similar manner to glycine. It is revealed that irradiation at the air/solution interface of the solution droplet much improved the crystallization probability, compared with that of fs laser irradiation inside the solution and that of spontaneous crystallization. X-ray diffraction analysis of the obtained crystals clarified that crystal structure was the same under each crystallization condition. It is known that protein can be adsorbed and be localized at the solution surface leading to the denaturation. However, this result indicates that the salting-out effect can stabilize the structure even at the surface, which is contemporarily reported in elsewhere. The present result clearly shows that utilizing solution surface irradiation in the fs laser-induced crystallization sharpens the existent crystallization technique. Further spectroscopic dynamics study on the early stage of molecular nucleation occurring at the surface is promising to establish novel interface/surface molecular science from this crystallization methodology.