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抄録
コロイド滴定法によるリポソームの表面荷電の定量について検討を行った.正コロイド試薬としてポリジアリルジメチルアンモニウムクロライドを,負コロイド試薬としてポリビニル硫酸カリウムを用いた.リポソームは,精製したリン脂質を超音波照射によって作製した.滴定の結果,ホスファチジルコリンは同一分子内にあるリン酸基と第四級アミンとの分子内の強い塩形成のために,pH2〜11の全領域にわたって荷電0となった.ホスファチジルグリセロールはpH3以上では常に一定負荷電量,0.633×10^<-6>当量/10^<-6>総リン当量を示した.ホスファチジルエタノールアミンは,低いpHではリン酸基とアミノ基が塩形成をしているため荷電0であるが,-NH_3^+→-NH_2への変化に伴って塩形成がこわれてリン酸基が正コロイドと結合するようになり,pH10.5以上では一定負荷電量,0.612×10^<-6>当量/10^<-6>総リン当量が得られた.ホスファチジルセリンではpH6〜7及びpH10.5以上で,各々0.541及び1.087×10^<-6>当量/10^<-6>総リン当量の一定負荷電量をもつ二段階の解離がみられた.両者は各々,カルボキシル基及びリン酸基の荷電量に対応する.又,このことからカルボキシル基:リン酸基:アミノ基の構成比が1:1:1であることも示された.本法では試料量10〜20mgでpH2〜11までの広いpH領域にわたる滴定が可能であり,官能基の解離状態の検索並びに荷電定量が行える.独立2回以上の実験結果から再現性は良好であることが分かった.
Surface charge on the liposome formed from purified phospholipids was determined by colloidal microtitration with potassium polyvinylsulfate (PVSK) and polydiallydimethylammonium chloride (Cat-Floe). The end point was detected by the color change of 0.1% TB from blue to red-violet. A micro colloidal titration apparatus (Gilmont ultra-precision micrometer syringe, No. S3200) was used for titration. Phospholipids used in this experiment were as folows : phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylserine (PS). The chloroform solution of the phospholipid was transferred into a 25-ml stoppered teat tube and evaporated to dryness in vacuo at 35〜36℃. The thin film of the phospholipid in the vessel was suspended in distilled water (20mg of lipid/2ml) by sonicating for 30min at room temperature. Stability of the liposome, whcih is a closed system composed of the lipid bilayer, was checked by using liposome entrapping glucose. From the results of colloidal titration, the surface of PC liposome was found not to be charged over the pH range from 2 to 11. This is considered due to formation of the intramolecular salt linkage between the phosphate group (pK_a =⃥2) and quarternary ammonium group of choline (pK_a =⃥13). PG liposome was negatively charged at above pH 3 with a constant value, (0.633±0.020)×10^<-6> eq/10^<-6> eq of phosphorus. The plots of the equivalent of negative charge against the amount of phospholipid gave a straight line. The results of three separate determinations indicated good reproducibility. PE liposome showed a constant value, 0.646×lO^<-6> eq/10^<-6> eq of phosphorus, at above pH 10.5. In the case of PS liposome, the constituent ratio of the functional groups was well corelated to the results of colloidal titration. From the constant values in the pH range of 6 to 7 and 10 to 11, the amounts of negative charge of the carboxyl and phosphate groups were determined as 0.541 and 0.546×lO^<-6> eq/10^<-6> eq of phosphorus, respectively. This negative charge of the liposome was considered to reflect the surface charge of the closed vesicle of the bilayer of the phospholipids. Colloidal micro-titration is useful not only for detecting the state of the functional groups over a wide pH range, but also for quantitative determination of the negative charge on the surface of a small amount (0.5 to 1.0mg of lipid/sample) of the phospholipid liposome.
