Duerkop Mark, Berger Eva, Dürauer Astrid, Jungbauer Alois
Austrian Centre of Industrial Biotechnology Continuous Integrated Manufacturing Vienna Austria.
University of Natural Resources and Life Sciences Department of Biotechnology Vienna Austria.
Eng Life Sci. 2018 Mar;18(3):169-178. doi: 10.1002/elsc.201700079. Epub 2017 Dec 4.
Neither the influence of high shear rates nor the impact of cavitation on protein aggregation is fully understood. The effect of cavitation bubble collapse-derived hydroxyl radicals on the aggregation behavior of human serum albumin (HSA) was investigated. Radicals were generated by pumping through a micro-orifice, ultra-sonication, or chemically by Fenton's reaction. The amount of radicals produced by the two mechanical methods (0.12 and 11.25 nmol/(L min)) was not enough to change the protein integrity. In contrast, Fenton's reaction resulted in 382 nmol/(L min) of radicals, inducing protein aggregation. However, the micro-orifice promoted the formation of soluble dimeric HSA aggregates. A validated computational fluid dynamic model of the orifice revealed a maximum and average shear rate on the order of 10 s and 1.2 × 10 s, respectively. Although these values are among the highest ever reported in the literature, dimer formation did not occur when we used the same flow rate but suppressed cavitation. Therefore, aggregation is most likely caused by the increased surface area due to cavitation-mediated bubble growth, not by hydroxyl radical release or shear stress as often reported.
高剪切速率的影响以及空化对蛋白质聚集的影响都尚未完全了解。研究了空化气泡坍塌产生的羟基自由基对人血清白蛋白（HSA）聚集行为的影响。自由基通过微孔隙泵送、超声处理或通过芬顿反应化学产生。两种机械方法产生的自由基量（0.12和11.25 nmol/（L·min））不足以改变蛋白质的完整性。相比之下，芬顿反应产生了382 nmol/（L·min）的自由基，诱导了蛋白质聚集。然而，微孔隙促进了可溶性二聚体HSA聚集体的形成。经过验证的孔隙计算流体动力学模型显示，最大和平均剪切速率分别约为10⁵ s⁻¹和1.2×10⁴ s⁻¹。尽管这些值是文献中报道过的最高值之一，但当我们使用相同流速但抑制空化时，并未发生二聚体形成。因此，聚集很可能是由空化介导的气泡生长导致的表面积增加引起的，而不是如通常报道的那样由羟基自由基释放或剪切应力引起的。
