Shoukry M I, Gong E L, Nichols A V
Lawrence Berkeley Laboratory, University of California, Donner Laboratory, Berkeley 94720.
Biochim Biophys Acta. 1994 Jan 20;1210(3):355-60. doi: 10.1016/0005-2760(94)90240-2.
We investigated the effect of Cu2+ catalyzed peroxidation on the status of tryptophan (Trp) in protein moieties in HDL and LDL together with its effect on apolipoprotein-lipid association. Incubation of HDL with Cu2+ resulted in a rapid decrease of Trp fluorescence intensity with time with a concomitant increase in Trp maximum emission wavelength (lambda max). LDL incubated with Cu2+ also showed a rapid decrease in Trp fluorescence intensity with time, with no associated increase in lambda max. The status of apo HDL and apo LDL was investigated after 4 h oxidation (4h-oxHDL and 4h-oxLDL respectively). With 4h-oxHDL, the shift in lambda max was not associated with protein dissociation but rather with protein crosslinking and formation of larger HDL species. Progressive increase in lambda max was observed in 4h-oxHDL with increase in guanidine hydrochloride (GuHCl) concentration; this was not due to protein dissociation. Although oxidation of LDL did not produce an increase in lambda max, a significant increase in wavelength was observed when 4h-oxLDL was exposed to increasing concentration of GuHCl. SDS-polyacrylamide gel electrophoresis and nondenaturing gradient gel electrophoresis of the 4h-oxLDL indicated formation of smaller molecular weight protein fragments that were still associated with LDL. Ultracentrifugation of oxidized LDL in the presence and absence of GuHCl showed no dissociated protein. In summary, these data indicate the following: (a) lipid peroxidation has a direct effect on Trp residues in both HDL and LDL, (b) oxidation of HDL is associated with conformational change in apo HDL, crosslinking and formation of larger particles, (c) oxidized HDL have a more stable apolipoprotein-lipid association than native HDL, (d) oxidation of LDL is associated with changes in apo B, that by fluorescence are apparent only in presence of GuHCl and results in fragmentation of apo B without dissociation of protein or change in particle size, and (e) stability of apolipoprotein-lipid association is comparable in oxidized and native LDL.
我们研究了Cu2+催化的过氧化作用对高密度脂蛋白（HDL）和低密度脂蛋白（LDL）中蛋白质部分色氨酸（Trp）状态的影响，以及其对载脂蛋白 - 脂质结合的影响。HDL与Cu2+孵育导致Trp荧光强度随时间迅速降低，同时Trp最大发射波长（λmax）增加。LDL与Cu2+孵育也显示Trp荧光强度随时间迅速降低，但λmax没有相关增加。在氧化4小时后（分别为4小时氧化的HDL和4小时氧化的LDL）研究了载脂蛋白HDL和载脂蛋白LDL的状态。对于4小时氧化的HDL，λmax的变化与蛋白质解离无关，而是与蛋白质交联和形成更大的HDL种类有关。在4小时氧化的HDL中，随着盐酸胍（GuHCl）浓度的增加，观察到λmax逐渐增加；这不是由于蛋白质解离。虽然LDL的氧化没有导致λmax增加，但当4小时氧化的LDL暴露于浓度不断增加的GuHCl时，观察到波长有显著增加。4小时氧化的LDL的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和非变性梯度凝胶电泳表明形成了仍与LDL相关的较小分子量蛋白质片段。在有和没有GuHCl的情况下对氧化的LDL进行超速离心，未显示出解离的蛋白质。总之，这些数据表明：（a）脂质过氧化对HDL和LDL中的Trp残基有直接影响，（b）HDL的氧化与载脂蛋白HDL的构象变化、交联和更大颗粒的形成有关，（c）氧化的HDL比天然HDL具有更稳定的载脂蛋白 - 脂质结合，（d）LDL的氧化与载脂蛋白B的变化有关，通过荧光仅在存在GuHCl时才明显，并导致载脂蛋白B碎片化，而蛋白质不解离或颗粒大小不变，以及（e）氧化的和天然的LDL中载脂蛋白 - 脂质结合的稳定性相当。
