DECOUPLING OF MELTING DOMAINS IN IMMOBILIZED RIBONUCLEASE-A

Ribonuclease A has been immobilized on silica beads through glutaralde yde-mediated chemical coupling in order to improve the stability of th e protein against thermal denaturation. The thermodynamic and binding properties of the immobilized enzyme have been studied and compared wi th those of the free enzyme. The parameters describing the binding of the inhibitor 3'-CMP (K-a and Delta H) as monitored by spectrophotomet ry and calorimetry were not significantly affected after immobilizatio n. Conversely both the stability and unfolding mechanism drastically c hanged. Thermodynamic analysis of the DSC data suggests that uncouplin g of protein domains has occurred as a consequence of the immobilizati on. The two state approximation of the protein unfolding process is no t longer valid for the immobilized RNase. Protein stability strongly d epends on the hydrophobicity properties of the support surface as well as on the presence of the inhibitor and pH. For example, after immobi lization on a highly hydrophobic surface, the enzyme is partially in t he unfolded state. The binding of a ligand is able to reorganize the p rotein structure into a native-like conformation. The refolding rates are different for the two protein domains and vary as a function of pH and presence of the inhibitor 3'-CMP. (C) 1994 Wiley-Liss, Inc.