High-performance gel-permeation chromatographic analysis of protein aggregation. Application to bovine carbonic anhydrase.

Protein association in bovine carbonic anhydrase (bCA) was investigated by high-performance gel-permeation chromatography (HPGPC). It was shown that bCA undergoes a time-dependent aggregation after freezing and storing at -20 degrees C. The addition of 50% ethylene glycol or glycerol prevented the aggregation. The HPGPC data were confirmed by the use of cross-linking with subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoretic analysis. The esterase activity of the aggregated bCA was comparable to that of the monomer, suggesting that the observed protein association does not affect the esterase catalytic centre of bCA. This study illustrates how HPGPC can be a convenient and reliable tool for monitoring storage-induced aggregation of proteins.