Ortwerth B J, Olesen P R
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Biochim Biophys Acta. 1988 Aug 31;956(1):10-22. doi: 10.1016/0167-4838(88)90292-0.
The incubation of calf lens extracts with 20 mM ascorbic acid under sterile conditions for 8 weeks caused extensive protein crosslinking, which was not observed with either 20 mM sorbitol or 20 mM glucose. While no precipitation was observed, ascorbic acid did induce the formation of high-molecular-weight protein aggregates as determined by Agarose A-5m chromatography. Proteins modified by ascorbic acid bound strongly to a boronate affinity column, however, crosslinked proteins were present mainly in the unbound fraction. These observations suggest that the cis-diol groups of ascorbic acid were present in the primary adduct, but were either lost during the crosslinking reaction or sterically hindered from binding to the column matrix. The amino acid composition of the ascorbic acid-modified proteins was identical to controls except for a 15% decrease in lysine. Amino acid analysis after borohydride reduction, however, showed a 25% decrease in lysine, a 7% decrease in arginine and an additional peak which eluted between phenylalanine and histidine. Extensive browning occurred during the ascorbic acid-modification reaction. This resulted in protein-bound chromophores with a broad absorption spectrum from 300 to 400 nm, and protein-bound fluorophores with excitation/emission maxima of 350/450 nm. A 4 week incubation of dialyzed crude lens extract with [1-14C]ascorbic acid showed increased incorporation for 2 weeks, followed by a decrease over the next 2 weeks as crosslinking was initiated. The addition of cyanoborohydride to the reaction mixture completely inhibited crosslinking and increased [1-14C]ascorbic acid incorporation to a plateau value of 180 nmol per mg protein. Amino acid analysis showed a 50% loss of lysine, and 8% decrease in arginine and the presence of a new peak which eluted slightly earlier than methionine. These data are consistent with the non-enzymatic glycation of lens proteins by either ascorbic acid or an oxidation product of ascorbic acid via a Maillard-type reaction.
在无菌条件下，将小牛晶状体提取物与20 mM抗坏血酸孵育8周会导致广泛的蛋白质交联，而20 mM山梨醇或20 mM葡萄糖则不会出现这种情况。虽然未观察到沉淀现象，但通过琼脂糖A - 5m色谱法测定，抗坏血酸确实诱导了高分子量蛋白质聚集体的形成。经抗坏血酸修饰的蛋白质与硼酸酯亲和柱强烈结合，然而，交联蛋白主要存在于未结合部分。这些观察结果表明，抗坏血酸的顺式二醇基团存在于初级加合物中，但在交联反应过程中要么丢失，要么在空间上受阻而无法与柱基质结合。除赖氨酸减少15%外，抗坏血酸修饰的蛋白质的氨基酸组成与对照相同。然而，硼氢化钠还原后的氨基酸分析显示，赖氨酸减少了25%，精氨酸减少了7%，并且在苯丙氨酸和组氨酸之间出现了一个额外的峰。在抗坏血酸修饰反应过程中发生了广泛的褐变。这导致蛋白质结合的发色团在300至400 nm范围内具有宽吸收光谱，以及蛋白质结合的荧光团，其激发/发射最大值为350/450 nm。用[1 - 14C]抗坏血酸对透析后的晶状体粗提物进行4周孵育，结果显示在2周内掺入量增加，随后在接下来的2周内随着交联的开始而减少。向反应混合物中加入氰基硼氢化钠完全抑制了交联，并使[1 - 14C]抗坏血酸掺入量增加到每毫克蛋白质180 nmol的稳定值。氨基酸分析显示赖氨酸损失了50%，精氨酸减少了8%，并且出现了一个比蛋氨酸洗脱稍早的新峰。这些数据与抗坏血酸或抗坏血酸的氧化产物通过美拉德型反应对晶状体蛋白质进行非酶糖基化一致。
