Partial unfolding of carbonic anhydrase provides a method for its immobilization on hydrophobic adsorbents and protects it against irreversible thermoinactivation

Our previous studies indicated that native carbonic anhydrase does not interact with hydrophobic adsorbents and that it acquires this ability upon denaturation. It was concluded that hydrophobic sites become available and that catalytically active immobilized preparations may be finally obtained. In the present study, alkyl-substituted Sepharose 4B containing octyl, dodecyl or palmityl residues (Sepharose-lipid) were used for adsorptive immobilization of heat-denatured forms of this protein. The enzyme adsorbed on octyl-Sepharose showed the highest kcat/Km(app) and the greatest thermostability as compared to the other two preparations. Moreover, the pH stability and resistance to the effect of KSCN, as a chaotropic salt, was higher for this preparation of the immobilized enzyme. Thermal stability and aggregation studies suggested that the enzyme is protected against irreversible thermoinactivation by interaction with the alkyl residues upon adsorption. Proline prevented both aggregation of the free enzyme at elevated temperatures and its interaction with the hydrophobic support. The higher degree of substitution in octyl-Sepharose appears to provide it with a greater capacity for multipoint interaction. It is suggested that the type of studies presented may provide useful information in connection with formation of intermediate states, in addition to its importance related to protein immobilization by adsorption.