Single GUV Method Reveals Interaction of Tea Catechin (-)-Epigallocatechin Gallate with Lipid Membranes
Yukihiro Tamba 1, Shinya Ohba 1, Masayo Kubota 1, Hiroe Yoshioka 1, Hisashi Yoshioka 2 and Masahito Yamazaki 3*
1 Shizuoka University
2 University of Shizuoka
3 Shizuoka University, Graduate School of Science and Technology
* To whom correspondence should be addressed. E-mail: spmyama{at}ipc.shizuoka.ac.jp.
Submitted on September 8, 2006
Revised on November 16, 2006
Accepted on 4 January 2007
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Abstract |
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Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In the present study, we investigated the interaction of (-)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC), using phase-contrast fluorescence microscopy and the single GUV method.
We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration (~150 mM NaCl) using the PEG-lipid method. Low concentrations of EGCg (above 30 micro-M) induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUV on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time, and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small-angle X-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles
Key Words:
bursting of liposomes, catechins, giant liposome, leakage, single GUV method
