Probing of Lipase Activity at Air/Water Interface by Sum-Frequency Generation Spectroscopy
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- 作者：Gediminas Niaura ; Zenonas Kuprionis ; Ilja Ignatjev ; Maryt Kaemkait ; Gintaras Valincius ; Zita Talaikyt ; Valdemaras Razumas ; Allan Svendsen
- 刊名：Journal of Physical Chemistry B
- 出版年：2008
- 出版时间：April 3, 2008
- 年：2008
- 卷：112
- 期：13
- 页码：4094 - 4101
- 全文大小：169K
- 年卷期：v.112,no.13(April 3, 2008)
- ISSN：1520-5207
文摘
The infrared-visible sum-frequency generation (SFG) vibrational spectroscopy was used to probe enzymaticactivity of Thermomyces lanuginosus lipase (TLL) at air/water interface. A monolayer of amphiphilicO-palmitoyl-2,3-dicyanohydroquinone (PDCHQ), containing target ester group and two CN groups servingas vibrational markers, was utilized as an enzyme substrate. SFG data revealed the detailed molecular scalestructure and properties of the PDCHQ layer at the interface. In particular, we demonstrate that hydrophilicheadgroup of PDCHQ is mainly in the form of an oxyanion, and the enzyme-induced cleavage of the esterbond could be spectroscopically monitored by the disappearance of the intense C
N resonance at 2224 cm-1.The enzymatic nature of the ester bond cleavage was confirmed by the control experiments with deactivatedS146A mutant variant of TLL. By comparing action of wild type (WT) TLL and its inactive S146A mutant,it was shown that two effects take place at the interface: disordering of the lipid monolayer due to the adsorptionof enzyme and enzymatic cleavage of the ester bond. The concentration of enzyme as low as 10 nM couldbe easily sensed by the SFG spectroscopy. We present spectroscopic evidence that upon hydrolysis one ofthe products, 2,3-dicyanohydroquinone, leaves the surface, while the other, palmitic acid, remains at air/water interface in predominantly undissociated form with the mono-hydrogen-bonded carbonyl group. Strongamide I (1662 cm-1) and amide A (3320 cm-1) SFG signals from TLL suggest that enzyme molecules positionthemselves at air/water interface in an orderly fashion. Presented work demonstrates the potential of SFGspectroscopy for in situ real-time monitoring of enzymatic processes at air/water interface.
N resonance at 2224 cm-1.The enzymatic nature of the ester bond cleavage was confirmed by the control experiments with deactivatedS146A mutant variant of TLL. By comparing action of wild type (WT) TLL and its inactive S146A mutant,it was shown that two effects take place at the interface: disordering of the lipid monolayer due to the adsorptionof enzyme and enzymatic cleavage of the ester bond. The concentration of enzyme as low as 10 nM couldbe easily sensed by the SFG spectroscopy. We present spectroscopic evidence that upon hydrolysis one ofthe products, 2,3-dicyanohydroquinone, leaves the surface, while the other, palmitic acid, remains at air/water interface in predominantly undissociated form with the mono-hydrogen-bonded carbonyl group. Strongamide I (1662 cm-1) and amide A (3320 cm-1) SFG signals from TLL suggest that enzyme molecules positionthemselves at air/water interface in an orderly fashion. Presented work demonstrates the potential of SFGspectroscopy for in situ real-time monitoring of enzymatic processes at air/water interface.