Interfacial behaviour of bovine testis hyaluronidase.
The interfacial properties of bovine testicular hyaluronidase were investigated 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. In whichever way the interfacial films were obtained (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 onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a 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 Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed 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, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.
Belem-Gonçalves
Silvia
S
Laboratoire Organisation et Dynamique des Membranes Biologiques, UMR-CNRS 5013, Université Claude Bernard Lyon I, 43, boulevard du 11 novembre 1918, F-69622 Villeurbanne Cedex, France.
Tsan
Pascale
P
Lancelin
Jean-Marc
JM
Alves
Tito L M
TL
Salim
Vera M
VM
Besson
Françoise
F
D000327:Adsorption; D000388:Air; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002021:Buffers; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013696:Temperature; D013737:Testis / Q000201:enzymology*; D014867:Water
D002021:Buffers; D014867:Water; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase