Having worked with bovine carbonic anhydrase (BCA) in several studies, including studies attempting to block its aggregation through ANS, examine its porosity through ANS binding, and explore the efficacy of phage-display-derived peptides to interfere with its aggregation, we wished to next explore whether BCA aggregates happen to contain large sections of the BCA chain retained in native structural format within the aggregates. Briefly, we wished to determine whether sub-domains of BCA’s structure were retained within thermally-derived aggregates of BCA is native format owing to aggregation resulting from only some partial disassociation of such sub-domains of structure, away from each other. We used proteolysis by a non-specific protease, subtilisin, to examine exposed peptide bonds in native and aggregated BCA which get cleaved, without the consequent (and subsequent) cleavage of any further peptide bonds within the sub-domain liberated by the proteolysis. We examined this through SDS-PAGE electrophoresis, to separate sub-domains of BCA liberated through limited and incomplete proteolysis. We performed N-terminal sequencing of the SDS-PAGE bands, and located the peptide bonds immediately preceding the N-terminal residues of fragments on the structural model of BCA. We discovered that, both in native and thermally-aggregated BCA, the cleavages were occurring at (or close) to peptide bonds which were clearly exposed to the solvent on the surface of BCA in the native structure, with no large side chains in the vicinity to protect the peptide bonds from attack by subtilisin. The fact that the same sites remained vulnerable in native and aggregated BCA suggested that BCA aggregates retain a considerable amount of native structure.

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