The activity of ribonuclease A (RNase A) during adsorption onto molecular smooth mica increases from 16% to 78% in a period of 24 h when compared to its activity in free solution at pH 5 and 20 + 0.5MC. From electropotential plots, the tertiary structure of RNase A, the characteristics of the mica surface, and direct measurements of the intermolecular forces between two adsorbed enzyme layers, a molecular explanation is offered for the changing activity with time.Initially, the RNase A molecules lie flat-on the mica with their smallest axis perpendicular to and their active site facing the surface. As adsorption proceeds, the molecules slowly reorient until at long times they lie end-on with their largest axis perpendicular to the surface and their active site partially exposed to the free solution. A translational diffusion process is indicated for the phase transition and molecular reorientation of the RNase A molecules.The propensity for proteins to adsorb onto solid surfaces depends on their molecular properties, including size, charge, and chemical and physical structure. It also depends on the surface characteristics of the solid, the nature of the interactions between the surface and the proteins, and the solubility of the proteins in the solution. Although much of the literature deals with the amount of protein adsorbed as a function of solution concentration (isotherms), attention has recently been given to the mechanisms of adsorption and the structure and stability of the adsorbed molecules (1).The activity of biocatalytic molecules such as enzymes at solution-solid interfaces depends on their structural stability (2). Solid surfaces are able to disrupt intramolecular disulfide and hydrogen bonds and hence the "breathing" structure of enzymes. Intense interactions with surfaces can denature proteins (3), whereas weak interactions often leave the protein conformationally stable.In this report, we present a molecular explanation for the changing activity of a well-known conformationally stable protein, bovine pancreas ribonuclease A (RNase A) during the adsorption period onto mica. The four disulfide bonds and a very strong hydrogen bonding network support the rigidity of RNase A (4). Electropotential plots and the well-known crystal structure of RNase A together with the charge condition of the mica surface at pH 5 and 20°C are used to determine the probable orientation of the enzyme molecules at solid-liquid interfaces. Direct measurements of the adsorption kinetics of RNase A onto two curved mica surfaces and ofthe intermolecular forces between two separate RNase A layers (each on a different surface) during the adsorption process are described and used to quantitatively estimate the amount of enzyme associated with the surface and the thickness of the adsorbed layers, respectively. The kinetics of the change in enzyme activity and molecular reorientation of the surface is followed.
MATERIALS AND METHODSMaterials. KCI salt was purchased from Aldrich and was of suprapure grade (99.99+%). RNase...