奈米銀對HIV-1蛋白酶之抑制效果與交互作用

Inhibitory Effect and Interaction between Silver Nanoparticle and HIV-1 Protease

邢忠彥(Chung-Yen Shing)

指導教授 ： 李篤中

國立臺灣大學/工學院/化學工程學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02150

摘要

為發現不同於基於結構設計的新型蛋白酶抑制劑，本研究以自行合成的奈米銀粒子，測試對HIV-1蛋白酶的抑制效果，另外，為提出可能的奈米銀抑制機制，則使用螢光分析技術探討奈米銀與HIV-1蛋白酶在水溶液的交互作用。
實驗結果顯示，以聚乙烯吡咯烷酮(PVP)和檸檬酸根做穩定劑的膠體奈米銀(1-3 nm)皆為良好的蛋白酶抑制劑。在銀/蛋白酶的莫耳比為500、37 ℃、pH 7.0的條件下，與HIV-1蛋白酶控制組相比，PVP穩定跟檸檬酸根穩定的奈米銀能將蛋白酶的酵素最大反應速率(Vmax)由原先的250 μM/min，分別抑制為8.117與2.078 μM/min。由於抑制劑造成HIV-1蛋白酶的米氏常數(Km)與Vmax減低，故奈米銀的抑制類型較有可能屬於反競爭抑制。
在相對活性試驗中，本研究使用檸檬酸根穩定的奈米銀(2, 10, 35 nm)，以探討銀粒徑對抑制效果的影響。結果顯示在銀/蛋白酶的莫耳比大於250、37 ℃、pH 7.0的條件下，較小奈米銀(2,10 nm)可抑制HIV-1蛋白酶活性至10%。而對較大奈米銀(35 nm)而言，其抑制活性維持在30%且不隨銀/蛋白酶的莫耳比變化。
螢光分析結果顯示，檸檬酸根穩定的奈米銀是HIV-1蛋白酶的消光劑，其消光類型屬於靜態消光，表示奈米銀與蛋白酶結合、形成一吸收螢光放射的複合物。37 ℃下，奈米銀對蛋白酶的結合位點數為1個。由不同溫度下的結合常數，複合物形成反應的標準焓與熵分別為-96.91 kJ/mol與-170.0 J/mol/K。最後，利用螢光共振能量轉移理論(FRET)，可求得奈米銀結合位點與HIV-1蛋白酶上色氨酸的距離，37 ℃下，為5.05 nm。

關鍵字

奈米銀粒子 ； HIV-1蛋白酶酵素抑制劑 ； 螢光分析

並列摘要

To find a new kind PI different from other structure-based PIs, synthesized silver nanoparticles was examined in their inhibition activities against HIV-1 PR. Besides, the interaction between silver nanoparticles and HIV-1 PR in aqueous solution was also investigated with fluorimetric analysis to propose a possible inhibition mechanism.
The experiment result have shown that both Polyvinylpyrrolidone (PVP)-stabilized and citrate-stabilized colloidal silver nanoparticles (1-3 nm) were good PIs. Comparing with the maximum enzyme reaction rate (Vmax) of HIV-1 PR control (250 μM/min), these two kinds of silver nanoparticles reduced Vmax to 8.117, 2.078 μM/min respectively with Ag/PR molar ratio of 500 at 37 ℃, pH 7.0. The type of both silver nanoparticles inhibition was suggested to be uncompetitive by the reduced Michaelis–Menten constant (Km) and Vmax.
Citrate-stabilized silver nanoparticle with different sizes (2, 10, 35 nm) were exploited in relative activity assay of HIV-1 PR to investigate the NPs size effect on the inhibition activity. For smaller silver nanoparticles (2,10 nm), the relative activity of HIV-1 PR was inhibited to ~10% the activity of control with Ag/PR molar ratio greater than 250 at 37 ℃, pH 7.0. For larger silver nanoparticles (35 nm), the relative activity was inhibited to 30% and independent of Ag/PR molar ratio at 37 ℃, pH 7.0.
The fluorimetric analysis has shown that the fluorescence quenching of HIV-1 PR by citrate-stabilized silver nanoparticles was static quenching, indicating the silver nanoparticle bind with HIV-1 PR and formed a complex. The number of binding site was equal to 1 at 37 ℃. By using binding constant under different temperatures, the ΔH0 and ΔS0 of binding reaction were calculated to be -96.91 kJ/mol and -170.0 J/mol/K respectively. Finally, based on Fluorescence resonance energy transfer (FRET) theory, the distance between tryptophan residues on HIV-1 PR and silver nanoparticle binding site (37 ℃) is 5.05 nm.

並列關鍵字

Silver nanoparticle ； HIV-1 protease ； Enzyme inhibitior ； Fluorimetric analysis

參考文獻

Abou El-Nour, K. M., A. Eftaiha, A. Al-Warthan and R. A. Ammar (2010). "Synthesis and applications of silver nanoparticles." Arabian Journal of Chemistry 3(3): 135-140.

Albani, J. R. (2008). Principles and applications of fluorescence spectroscopy, Wiley-Blackwell.

Alcala, J. R., E. Gratton and F. Prendergast (1987). "Fluorescence lifetime distributions in proteins." Biophysical journal 51(4): 597-604.

Alt, V., T. Bechert, P. Steinrucke, M. Wagener, P. Seidel, E. Dingeldein, E. Domann and R. Schnettler (2004). "An in vitro assessment of the antibacterial properties and cytotoxicity of nanoparticulate silver bone cement." Biomaterials 25(18): 4383-4391.

Bonnemann, H., W. Brijoux, R. Brinkmann, R. Fretzen, T. Joussen, R. Koppler, B. Korall, P. Neiteler and J. Richter (1994). "Preparation, characterization, and application of fine metal particles and metal colloids using hydrotriorganoborates." Journal of molecular catalysis 86(1): 129-177.