Davies K J, Lin S W, Pacifici R E
J Biol Chem. 1987 Jul 15;262(20):9914-20.
Proteolytic degradation of oxidatively damaged [3H] bovine serum albumin [( 3H]BSA) was studied during incubation with cell-free erythrocyte extracts and a wide variety (14) of purified proteases. [3H]BSA was pretreated by exposure (60Co radiation) to the hydroxyl radical (.OH), the superoxide anion radical (O2-), or the combination of .OH + O2- + oxygen. Treated (and untreated) samples were dialyzed and then incubated with erythrocyte extract or proteases for measurements of proteolytic susceptibility (release of acid-soluble counts). Both .OH and .OH + O2- + caused severalfold increases in proteolytic susceptibility (with extract and proteases), but O2- alone had no effect. Proteolytic susceptibility reached a maximum at 15 nmol of .OH/nmol of BSA and declined thereafter. In contrast, proteolytic susceptibility was still increasing at an .OH + O2-/BSA molar ratio of 100 (50% .OH + 50% O2-). Degradation in erythrocyte extracts was conducted by a novel ATP- and Ca2+-independent pathway, with maximal activity at pH 7.8. Inhibitor profiles indicate that this pathway may involve metalloproteases and serine proteases. Comparisons of proteolytic susceptibility with multiple modifications to BSA primary, secondary, and tertiary structure revealed a high correlation (r = 0.98) with denaturation/increased hydrophobicity by low concentrations of .OH. Covalent aggregation reactions (BSA cross-linking) may explain the declining proteolytic susceptibility observed at .OH/BSA molar ratios greater than 20. Protein denaturation may also have caused the increased proteolytic susceptibility induced by .OH + O2- + O2, but no simple correlation could be obtained. Results with .OH + O2- + O2 appear to have been complicated by direct BSA fragmentation reactions involving (.OH-induced) protein radicals and oxygen. These data indicate a direct and quantitative relationship between protein damage by oxygen radicals and increased proteolytic susceptibility. Oxidative denaturation may exemplify a simple, yet effective inherent mechanism for intracellular proteolysis.
在与无细胞红细胞提取物及14种纯化蛋白酶共同孵育的过程中，对氧化损伤的[3H]牛血清白蛋白（[3H]BSA）的蛋白水解降解进行了研究。[3H]BSA通过暴露于（60Co辐射）羟基自由基（·OH）、超氧阴离子自由基（O2-）或·OH + O2- + 氧气的组合中进行预处理。将处理过的（和未处理的）样品进行透析，然后与红细胞提取物或蛋白酶一起孵育，以测量蛋白水解敏感性（酸溶性计数的释放）。·OH和·OH + O2- + 均使蛋白水解敏感性增加了几倍（与提取物和蛋白酶共同作用时），但单独的O2-没有影响。蛋白水解敏感性在·OH与BSA的摩尔比为15 nmol/ nmol时达到最大值，此后下降。相比之下，在·OH + O2-与BSA的摩尔比为100（50%·OH + 50% O2-）时，蛋白水解敏感性仍在增加。红细胞提取物中的降解是通过一种新的不依赖ATP和Ca2+的途径进行的，在pH 7.8时活性最高。抑制剂谱表明该途径可能涉及金属蛋白酶和丝氨酸蛋白酶。对BSA一级、二级和三级结构进行多种修饰后的蛋白水解敏感性比较显示，与低浓度·OH引起的变性/疏水性增加高度相关（r = 0.98）。共价聚集反应（BSA交联）可能解释了在·OH与BSA摩尔比大于20时观察到的蛋白水解敏感性下降。蛋白质变性也可能导致了·OH + O2- + O2引起的蛋白水解敏感性增加，但未获得简单的相关性。·OH + O2- + O2 的结果似乎因涉及（·OH诱导的）蛋白质自由基和氧气的直接BSA断裂反应而变得复杂。这些数据表明氧自由基对蛋白质的损伤与蛋白水解敏感性增加之间存在直接的定量关系。氧化变性可能是细胞内蛋白水解的一种简单而有效的内在机制的例证。
