Abstract
Small molecule colloidal aggregates adsorb and partially denature proteins, inhibiting them artifactually. Oddly, this inhibition is typically time-dependent. Two mechanisms might explain this: low concentrations of the colloid and enzyme might mean low encounter rates, or colloid-based protein denaturation might impose a kinetic barrier. These two mechanisms should have different concentration dependencies. Perplexingly, when enzyme concentration was increased, incubation times actually lengthened, inconsistent with both models and with classical chemical kinetics of solution species. We therefore considered molecular crowding, where colloids with lower protein surface density demand a shorter incubation time than more crowded colloids. To test this, we grew and shrank colloid surface area. As the surface area shrank, the incubation time lengthened, while as it increased, the converse was true. These observations support a crowding effect on protein binding to colloidal aggregates. Implications for drug delivery and for detecting aggregation-based inhibition will be discussed.