Abstract：
The ability of bovine serum albumin (BSA) to form condensates in crowded environments has been discovered only recently. Effects of this condensed state on the secondary structure of the protein have already been unraveled as some aging aspects, but the pseudo-enzymatic behavior of condensed BSA has never been reported yet. This article investigates the kinetic profile of para-nitrophenol acetate hydrolysis by BSA in its condensed state with poly(ethylene) glycol (PEG) as the crowding agent. Furthermore, the initial BSA concentration was varied between 0.25 and 1 mM which allowed us to modify the size distribution, the volume fraction, and the partition coefficient (varying from 136 to 180). Hence, the amount of BSA originally added was a simple way to modulate the size and density of the condensates. Compared with dilute BSA, the initial velocity (vi) with condensates was dramatically reduced. From the Michaelis-Menten fits, the extracted Michaelis constant Km and the maximum velocity Vmax decreased in control samples without condensates when the BSA concentration increased, which was attributed to BSA self-oligomerization. In samples containing condensates, the observed vi was interpreted as an effect of diluted BSA remaining in the supernatants and from the condensates. In supernatants, the crowding effect of PEG increased the kcat and catalytic efficiency. Last, Vmax was proportional to the volume fraction of the condensates, which could be controlled by varying its initial concentration. Hence, the major significance of this article is the control of the size and volume fraction of albumin condensates, along with their kinetic profile using liquid-liquid phase separation.
摘要：
牛血清白蛋白（BSA）在拥挤环境中形成冷凝物的能力最近才被发现。这种浓缩状态对蛋白质二级结构的影响已经被揭示为一些老化方面，但从未报道过凝聚态BSA的假酶行为。本文研究了以聚乙二醇（PEG）为拥挤剂，BSA在缩合状态下水解对硝基苯酚乙酸酯的动力学曲线。此外，初始BSA浓度在0.25和1mM之间变化，这允许我们修改大小分布，体积分数,和分配系数(从136变化到180)。因此，最初添加的BSA的量是调节冷凝物的大小和密度的简单方法。与稀释的BSA相比，冷凝物的初始速度（vi）急剧下降。从米迦勒-门顿适合，当BSA浓度增加时，在没有冷凝物的对照样品中，提取的米氏常数Km和最大速度Vmax降低，这归因于BSA自身寡聚化。在含有冷凝物的样品中，观察到的vi被解释为保留在上清液和冷凝物中的稀释的BSA的作用。在上清液中，PEG的拥挤效应提高了kcat和催化效率。最后,Vmax与冷凝物的体积分数成正比，可以通过改变其初始浓度来控制。因此，本文的主要意义是控制白蛋白缩合物的大小和体积分数，以及它们使用液-液相分离的动力学曲线。
