Interfacial behaviour of bovine testis hyaluronidase
Résumé
The interfacial properties of bovine testicular hyaluronidase were suggested by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. Whatever the way to obtain interfacial films (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein on the same subphase, indicating that bovine testicular hyaluronidase molecules adopted similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by FTIR spectroscopy, were discussed in relation with the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the hyaluronidase interfacial properties. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed the substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation with the modelled structure of bovine testis hyaluronidase where a hydrophobic region was supposed to be at the opposite of the catalytic site.
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