Kinetic Study of the Esterase-like Activity of Albumin following Condensation by Macromolecular Crowding

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 pseudoenzymatic 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 (v i ) with condensates was dramatically reduced. From the Michaelis−Menten fits, the extracted Michaelis constant K m and the maximum velocity V max decreased in control samples without condensates when the BSA concentration increased, which was attributed to BSA self-oligomerization. In samples containing condensates, the observed v i 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 k cat and catalytic efficiency. Last, V max 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.