We suspected that the heating-derived aggregates of bovine carbonic anhydrase (BCA) must be porous, and not internally solid and well-packed like the insides of any well-folded protein molecule (which is like a well-fitted three-dimensional jigsaw puzzle). To explore this possibility with BCA, using it as a representative of the thermal aggregates of any globular protein, we used the hydrophobicity-seeking dye ANS to show that such aggregates retain enough space within themselves to undergo unfolding and refolding of chain sections without disaggregation or reaggregation. Once thermally-derived aggregates of BCA are made, they do not either disaggregate upon cooling or reaggregate upon heating. Our question was, ‘what is happening within this once-made aggregates, when they are heated or cooled’? The answer we obtained, using ANS binding, was that (discounting for the loss of quantum yield of ANS with heating), there are voids within porous BCA aggregates in which chain sections are free to undergo pseudo-folding and unfolding reactions even while the aggregate undergoes no change in status. Pseudo-folding creates structures with unsatisfied hydrophobic surfaces which are assessed through ANS binding. The study demonstrates that aggregates can be porous to the free flow of ANS dye, and therefore, of any small molecule. Consequently, any designer aggregates made with enzymes that remain active after aggregation could function like immobilized enzymes, with substrates diffusing through them.

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