MODIFICATION OF PROTEIN BY THE LIPID PEROXIDATION PRODUCT 4-HYDROXY-2-NONENAL

活性氧容易与多不饱和脂肪酸相互作用 导致形成细胞毒性醛类，例如4-羟基-2-甲基-2-（4-羟基-2-甲基苯基）-N， 壬烯醛（HNE）和丙二醛。 在动物组织中，脂质过氧化 一些与衰老有关的退行性疾病的增加， 荧光物质被认为是由脂质 过氧化产物与蛋白质随年龄积累。 修改 蛋白质和其他生物分子的脂质过氧化产物， 因此被认为有助于衰老过程。 的机制 而几个潜在反应的相对贡献不是， 但是，这是很好理解。 来自明串珠菌的葡萄糖-6-磷酸脱氢酶（Glu-6-PDH） 肠系膜不含半胱氨酸残基，被选为 研究HNE与亲核试剂相互作用的简便模型 蛋白质中的胺 Glu-g-PDH与HNE的延长孵育导致 交联蛋白质的形成。 这是伴随着 蛋白质相关荧光的出现使人联想到脂褐素， 激发和发射最大值分别为340和415 nm。 交联蛋白质比天然Glu-6-PDH更不易受 通过多催化蛋白酶的蛋白水解，一种多酶的蛋白水解 参与受损蛋白质的细胞内降解的复合物。 此外，HNE修饰的Glu-6-PDH抑制多催化蛋白酶 因此可以防止氧化蛋白质的有效降解。 交联Glu-6-PDH的形成与近 仅损失赖氨酸残基。 使用色谱和质谱 光谱技术，我们发现赖氨酸的ε-氨基 与HNE的双键（C3）反应形成迈克尔加合物， 然后在羰基处与第二个赖氨酸残基反应， 部分（C1）以形成席夫碱交联。 的结构 荧光团目前正在研究中。 这些结果和 相关的实验可能会提供关键信息的作用，脂质 与疾病和年龄相关的受损蛋白质积累中的过氧化作用 和细胞功能受损。

Reactive oxygen species readily interact with polyunsaturated fatty acids resulting in the formation of cytotoxic aldehydes such as 4-hydroxy-2- nonenal (HNE) and malondialdehyde. In animal tissues, lipid peroxidation increases in a number of degenerative diseases associated with aging and fluorescent material believed to results from interaction of lipid peroxidation products with protein accumulates with age. Modification of protein and other biomolecules by lipid peroxidation products is therefore believed to contribute to the aging process. The mechanisms and relative contributions of several potential reactions are not, however, well understood. Glucose-6-phosphate dehydrogenase (Glu-6-PDH) from Leuconostoc mesenteroides contains no cysteine residues and was chosen as a convenient model for studying the interaction of HNE with nucleophilic amines in protein. Prolonged incubation of Glu-g-PDH with HNE leads to the formation of cross-linked protein. This is accompanied by the appearance of protein-associated fluorescence reminiscent of lipofuscin, with excitation and emission maxima of 340 and 415 nm, respectively. Cross-linked protein is less susceptible than native Glu-6-PDH to proteolysis by the multicatalytic protease, a multienzymic proteolytic complex involved in the intracellular degradation of damaged proteins. In addition, HNE-modified Glu-6-PDH inhibits the multicatalytic protease and can therefore prevent the efficient degradation of oxidized protein. Formation of cross-linked Glu-6-PDH is associated with the nearly exclusively loss of lysine residues. Using chromatographic and mass spectral techniques, we found that an epsilon-amino group of lysine reacts with the double bond (C3) of HNE to form a Michael adduct, which then undergoes reaction with a second lysine residue at the carbonyl moiety (C1) to form a Schiff base cross-link. The structure of the fluorophore is presently under investigation. The results of these and related experiments may provide critical information on the role of lipid peroxidation in disease- and age-related accumulation of damaged protein and impairment of cellular function.