利用螢光偵測技術探討蜂毒胜肽之膜穿孔行為

Abstract

蜂毒胜肽是蜜蜂 (Apis mellifera) 毒液中的主要成分，除了是溶血毒素之外，也有殺菌能力，因此也屬於抗菌胜肽。蜂毒胜肽會使細胞膜變薄，也可在細胞膜上形成孔洞，導致細胞內液外洩，進而殺死細胞。雖然蜂毒胜肽的研究已有多年，但其在膜上形成孔洞的方式與過程並不完全清楚。為此我們建立了一套共聚焦螢光偵測系統，透過量測螢光分子洩漏進脂質體造成之螢光強度變化，即時觀測胜肽攻擊單一脂質體的過程。我們發現加入蜂毒胜肽之後，脂質體內之螢光訊號出現許多間歇性但強度相近的突峰 (spike)，類似所謂「單分子行為」。經分析突峰上升高度(rise height)、上升時間 (rise time)、突峰間距 (rise interval) 與峰毒胜肽濃度的關係發現：當蜂毒胜肽濃度升高，突峰之上升高度呈現簡單倍數關係，且峰值間距變小，但是突峰的上升時間不變。從二維的螢光影像中，我們發現脂質體受到蜂毒胜肽攻擊時，螢光上升過程中會脂質體內部螢光皆呈現均勻分布。基於以上初步結果，我們提出三個蜂毒胜肽攻擊細胞膜的可能模型。此外有文獻報導峰毒胜肽可改變細胞膜之流動性，影響膜上維繫細胞存活的生理功能，而導致細胞死亡。我們也利用「螢光相關光譜技術」觀察脂質膜的流動情形。初步結果顯示，蜂毒胜肽吸附於脂質膜後的確會降低脂質膜的流動性；而且當胜肽分子在膜表面的密度達到飽和後，流動性則維持穩定。

Melittin, a small hemolytic peptide, is the major component of the venom of the European honey bee Apis mellifera. Melittin possesses also antimicrobial ability and this can also be regarded as an antimicrobial peptide. Earlier work shows that melittin can cause membrane thinning and form pores on membranes; the latter can lead to leakage of intracellular fluid causing cell death. Although there have been extensive studies on the pore forming mechanism of melittin, details of this process remains illusive especially as the concentration of melittin is comparable or below the threshold concentration of pore formation. To address this issue, we have developed a novel mean, based on confocal detection of fluorescence leakage of single vesicles, to investigate interaction between peptides and lipid membranes in real-time. Significantly, we observed occasional occurrence of spikes of comparable intensities, an observation that resembles single-molecules behavior. We carried out single-molecule analysis on rise-height, rise-time, and rise-interval of the time trace of those fluorescence spikes, and the preliminary results show that that the rise-height can be approximated as multiples of a unit number, and the rise-interval decreases as the concentration of melittin increases, while the rise-time remains constant. On the other hand, time-lapse confocal images do not exhibit spatial heterogeneity in the intensity distribution. We proposed three plausible models to account for these observations. We have also carried out fluidity measurements using fluorescence correlation spectroscopy (FCS), and found that the treatment of melittin on membranes caused a significant decrease on membrane fluidity, and the change of fluidity exhibits dosage-dependence, but becomes insignificant as the concentration of melittin exceeds some value. The results can be accounted with the formation of domains resulted from electrostatic interaction between adsorbed melittins and lipids.