Ortwerth B J, Coots A, James H L, Linetsky M
Mason Eye Institute, University of Missouri, Columbia, Missouri 65212, USA.
Arch Biochem Biophys. 1998 Mar 15;351(2):189-96. doi: 10.1006/abbi.1997.0549.
The oxidation products of ascorbic acid (AscH-) can rapidly glycate and crosslink lens proteins in vitro, producing fluorophores and browning products similar to those present in cataractous lenses. The accumulation of AscH- oxidation products, however, would largely be prevented by the millimolar levels of glutathione (GSH) present in human lens. Here we investigate whether protein aggregation could allow the oxidation of AscH- by UVA-induced reactive oxygen species in the presence of physiological levels of GSH. The metal-catalyzed oxidation of 1.0 mM AscH- by 50 microM Cu(II) was almost complete after 1 h, but no oxidation was seen in the presence of GSH concentrations as low as 0.5 mM. UVA irradiation of protein aggregates from human lens, which accumulated more than 2.0 mM singlet oxygen after 1 h, caused a 50-60% oxidation of 1.0 mM AscH-. The addition of 204 mM GSH, however, decreased AscH- oxidation by less than half, and 30% of the AscH- was oxidized even in the presence of 15 mM GSH. This diminished protection may be due, in part, to the ability of AscH-, but not GSH, to penetrate to the sites of singlet oxygen generation located within the protein. Consistent with this hypothesis, greater GSH protection was seen when a proteolytic digest of the human proteins was subjected to the same irradiation or when singlet oxygen was chemically generated from 3-(4-methyl-1-naphthyl)propionic acid endoperoxide (MNPAE) at 37 degrees C in the medium. The addition of 50 microM Cu(II) had no effect on the rate of degradation of dehydroascorbic acid (DHA). Singlet oxygen, either UVA- or MNPAE-generated, increased the rate of DHA loss. This secondary oxidation of DHA by singlet oxygen would allow the accumulation of AscH- oxidation products was not reducible by GSH. Therefore, the data presented here argue that the protein aggregation seen in older human lenses may permit oxidized AscH--induced crosslinking to occur even at physiological GSH levels.
抗坏血酸（AscH-）的氧化产物在体外能迅速使晶状体蛋白糖化并交联，产生荧光团和褐变产物，类似于白内障晶状体中存在的物质。然而，人晶状体中存在的毫摩尔水平的谷胱甘肽（GSH）在很大程度上可防止AscH-氧化产物的积累。在此，我们研究在生理水平的GSH存在下，蛋白质聚集是否会使AscH-被紫外线诱导的活性氧氧化。1.0 mM AscH-被50 microM Cu(II)进行金属催化氧化，1小时后几乎完全氧化，但在GSH浓度低至0.5 mM时未观察到氧化现象。对人晶状体的蛋白质聚集体进行紫外线照射，1小时后积累了超过2.0 mM的单线态氧，导致1.0 mM AscH-发生50 - 60%的氧化。然而，添加204 mM GSH使AscH-氧化减少不到一半，即使在存在15 mM GSH的情况下仍有30%的AscH-被氧化。这种保护作用减弱可能部分归因于AscH-而非GSH能够渗透到蛋白质内单线态氧产生的部位。与此假设一致，当对人蛋白质的蛋白水解消化物进行相同照射时，或者当在37摄氏度的培养基中由3 - (4 - 甲基 - 1 - 萘基)丙酸内过氧化物（MNPAE）化学产生单线态氧时，观察到了更强的GSH保护作用。添加50 microM Cu(II)对脱氢抗坏血酸（DHA）的降解速率没有影响。由紫外线或MNPAE产生的单线态氧增加了DHA损失的速率。DHA被单线态氧的这种二次氧化将导致AscH-氧化产物的积累，而GSH无法将其还原。因此，此处呈现的数据表明，在老年人晶状体中看到的蛋白质聚集可能允许即使在生理GSH水平下也会发生氧化的AscH-诱导的交联。
