Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Research Method of Fatty Acids Transfer between Phospholipid Model Membranes

인지질 모델막에서의 지방산 이동에 관한 연구 방법

  • 임병순 (공주대학교 식품영양학과) ;
  • 김혜경 (한서대학교 식품생물공학과) ;
  • 김을상 (단국대학교 식품영양학과)
  • Published : 1997.08.01
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Abstract

Direct measurement of the kinetics of free fatty acid transfer between phospholipid model membrane is technically limited by the rapid nature of the transfer process. Separation of membrane-bound fatty acid by centrifugation has shown that although the equilibrium distribution of free fatty acid is determined by this method, fatty acid transfer occurs too rapidly for accurate kinetic measurements. Recently fluorescence resonance energy transfer(FRET) assay has been developed to examine transfer of fatty acids between membranes. Donor membranes which has fluorescent fatty acid, anthroyloxy fatty acid(AOFA), is mixed with acceptor membranes which has non-interchangeable fluorescent quencher, nitrobenzo-xadiazol(NBD), using stopped flow apparatus. As the fluorescent fatty acids transfer from donor membrane to acceptor membrane, fluorescence intensity would be decreased and the rate and degree of fatty acid transfer can be analyzed. Fatty acid transfer between micelles is more complicated because of bile salt. Therefore in experiments with micelles, fluorescence self quenching assay is used. At high concentrations, a fluorophore tends to quench its own fluorescence causing a reduction in fluorescence intensity. Donor micelles contained self quenching concentrations of fluorophore and acceptor micelles had no fluorophore. Upon mixing of donor and acceptor micelles, the rate of transfer of the fluorophore from the donor to the acceptor was measured by monitoring the release in self quenching when its concentration in donor decreased over time.

세포막에서의 지방산 이동은 매우 빠르게 일어나므로 방사성 원소를 사용해서는 여러가지 단점이 있고, 정확한 이동속도 측정에도 어려움이 많았다 최근에 개발된 FRET assay는 형광성 물질과 형광성 물질을 상쇄시키는 quencher를 사용한 실험방법 이다. 이는 공명 에너지 이동의 원리를 이용한 것으로 형광광도계, stopped-flow장치를 사용하여 소수성 물질 이동을 직접 컴퓨터 모니터로 측정하는 방법으로 기존방법의 단점을 보완하였다. Donor막에는 형광성 표지를 붙인 지방산이 들어 있고 acceptor막에는 형광을 흡수하는 물질이 들어 있어서 형광성 지방산이 donor에서 acceptor로 이동하면 형광도가 감소하며, 시간에 따른 형광도 감소를 측정하여 지방산 이동속도를 측정하는 방법이다. 형광성 표지를 이용하여 소수성 물질 이동에 사용되는 또 다른 방법은 self-Quenching assay이다. 형광 물질의 농도가 높아지면 서로 형광을 흡수하는 성질을 이용한 방법으로 주로 micelle에서의 물질 이동에 많이 쓰인다. Donor micelle에는 높은 농도의 형광성 지방산이 들어 있고 acceptor micelle에는 형광성 지방산이 들어 있지 않을 때 형광성 지방산이 donor에서 acceptor로 이동하면 형광도가 증가하게 되고 시간에 따른 형광도 증가를 측정하는 방법이다.

Keywords

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