Stability and equilibria of promiscuous aggregates in high protein milieus
Stability and equilibria of promiscuous aggregates in high protein milieus
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DOI：
10.1039/b616314a
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发表时间：
2007-03-01
影响因子：
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通讯作者：
Shoichet, Brian K.
中科院分区：
文献类型：
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作者：
Coan, Kristin E. D.;Shoichet, Brian K.
在微摩尔浓度下，许多分子在水溶液中形成聚集体。在这种形式下，它们非特异性地抑制酶，导致酶测定中的假阳性“命中”，特别是在高通量筛选时。牛血清白蛋白（BSA）可以减弱这种抑制作用;这种作用的机制尚不清楚。在这里，我们提出的证据表明，BSA，溶菌酶，胰蛋白酶，防止抑制时，在每毫升浓度与聚集体的毫克孵育之前，添加监测酶。这些解决方案仍然含有聚集体的动态光散射（DLS），这表明抑制是防止饱和的聚集体，而不是破坏it.For大多数组合的聚集体和蛋白质，抑制不逆转，如果竞争蛋白质加入后，聚集体与监测酶孵育。在观察到适度逆转的一个例外中，DLS和流式细胞术表明该效应是由于聚集体的破坏。这些结果表明，聚集体结合的酶与游离酶不处于动态平衡，并且结合的酶不能被竞争蛋白质取代。为了进一步验证这一假设，我们将聚集体结合酶与特定的不可逆抑制剂一起孵育，然后用洗涤剂破坏聚集体。大多数酶的活性恢复聚集体破坏，表明没有修改的不可逆抑制剂。这些结果表明，酶被紧密地结合到聚集体上，以防止任何明显的解离，而且，聚集体在生理相关的蛋白质浓度下是稳定的。
At micromolar concentrations, many molecules form aggregates in aqueous solution. In this form, they inhibit enzymes non-specifically leading to false positive "hits" in enzyme assays, especially when screened in high-throughput. This inhibition can be attenuated by bovine serum albumin (BSA); the mechanism of this effect is not understood. Here we present evidence that BSA, lysozyme, and trypsin prevent inhibition when incubated at milligram per millilitre concentrations with aggregates prior to the addition of the monitored enzyme. These solutions still contained aggregates by dynamic light scattering (DLS), suggesting that inhibition is prevented by saturating the aggregate, rather than disrupting it. For most combinations of aggregate and protein, inhibition was not reversed if the competing protein was added after the incubation of aggregates with the monitored enzyme. In the one exception where modest reversal was observed, DLS and flow cytometry indicated that the effect was due to the disruption of aggregates. These results suggest that aggregate-bound enzyme is not in dynamic equilibrium with free enzyme and that bound enzyme cannot be displaced by a competing protein. To further test this hypothesis, we incubated aggregate-bound enzyme with a specific, irreversible inhibitor and then disrupted the aggregates with detergent. Most enzyme activity was restored on aggregate disruption, indicating no modification by the irreversible inhibitor. These results suggest that enzyme is bound to aggregate so tightly as to prevent any noticeable dissociation and that furthermore, aggregates are stable at physiologically relevant concentrations of protein.