Wang J Y, Suzuki K, Miyazawa T, Ueki T, Kouyama T
Biophysics Laboratory, Institute of Physical and Chemical Research, Wako, Saitama, Japan.
Arch Biochem Biophys. 1996 Jun 15;330(2):387-94. doi: 10.1006/abbi.1996.0266.
Motional properties of fluorescent substances produced by lipid peroxidation by a time-resolved fluorescence polarization technique were studied. When liposomes containing phosphatidylethanolamine (PE) and linoleic hydrocarbon chain were incubated at 37 degrees C, fluorophores absorbing maximally at 360 nm and emitting near 430 nm were produced. Their fluorescence anisotropy decay measured at 23 degrees C was fitted well with a sum of a fast relaxation and a time-independent residual term. With the increase of oxidation degree, the time constant of the relaxation term increased. This may be explained by alteration in the membrane structure or by modification of the fluorescent products themselves. Information on the location of the fluorescent products was obtained when their motional property was compared with those of various extrinsic probes that were incorporated at different positions of the lipid bilayer. It was found that the motional property of the fluorescent oxidation products is similar to that of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, a rod-shaped hydrophobic probe with a charged terminal. Other probes sensing the polar region or the hydrophobic region of the membrane were characterized by a lower order parameter. It is suggested that the fluorescent oxidation products have a polar moiety located at the membrane surface and attached to the amino group of PE while the tail part being buried in the hydrophobic region of the membrane. This picture is supported by fluorescence quenching experiments with the aqueous quencher Co2+. On the other hand, fluorophores produced by the reaction of malondialdehyde and PE suggested to have a chemical structure in which the angle between the absorption and emission dipole moments is very large. On the basis of these observations, the production pathway of fluorophores in oxidized membranes is discussed.
采用时间分辨荧光偏振技术研究了脂质过氧化产生的荧光物质的运动特性。当含有磷脂酰乙醇胺（PE）和亚油酸烃链的脂质体在37℃孵育时，会产生在360nm处有最大吸收且在430nm附近发射荧光的荧光团。在23℃下测量其荧光各向异性衰减，结果与快速弛豫和与时间无关的残余项之和拟合良好。随着氧化程度的增加，弛豫项的时间常数增大。这可能是由于膜结构的改变或荧光产物本身的修饰所致。当将荧光产物的运动特性与掺入脂质双层不同位置的各种外在探针的运动特性进行比较时，获得了有关荧光产物位置的信息。发现荧光氧化产物的运动特性与1-(4-三甲基铵苯基)-6-苯基-1,3,5-己三烯相似，后者是一种带有带电末端的棒状疏水探针。其他检测膜极性区域或疏水区域的探针的特征是序参数较低。提示荧光氧化产物具有位于膜表面并与PE氨基相连的极性部分，而尾部则埋在膜的疏水区域中。用淬灭剂Co2+进行的荧光淬灭实验支持了这一观点。另一方面，丙二醛与PE反应产生的荧光团表明其化学结构中吸收和发射偶极矩之间的夹角非常大。基于这些观察结果，讨论了氧化膜中荧光团的产生途径。
