Microcalorimetric Study On Displacement Adsorption Enthalpies Of Denatured RNase A On Hydrophobic Surface

The MicroDSC-Ⅲcalorimeter was used to measure the displacement adsorption enthalpy of Ribonuclease A (RNase A) which originally had been denatured by various concentrations of guanidine hydrochloride (GuHCl) mixing with moderately hydrophobic PEG packings in 1.5 mol.L-1 ammonium sulfate ((NH4)2SO4) salt solutions at 25℃. According to the△H which was divided into four enthalpy fractions by stoichiometric displacement theory (SDT) and adsorption isotherm data, the thermodynamic contribution of enthalpy fractions was discussed. The adsorption folding law also was analyzed by CD,FTIR and DSC.The thermal stability of adsorbed RNase A was decreased comparing with in the solution and adsorbed RNase A was more stable in higher surface coverage or higher GuHCl concentrations when the other factor is fixed. Conformational loss may occurred on adsorbed RNase A at zero and lower concentrations of GuHCl.The△H values increase first and then decrease with the increment of GuHCl concentration whether the concentrations of adsorbed RNase A was 0.4,0.7 or 1.0 mg.ml^-1. An exothermic effect for native RNase A is mainly attributed mainly to hydration associated with conformational loss, based on the thermal analysis of enthalpy fractions of protein adsorption, it was expressed asΔΗmh in the enthalpy fractions, while a great endothermic effect for 0.3 mol/L GuHCl is induced by conformational structure loss which was denotedΔΗml in the enthalpy fractions.The measured△H values of RNase A in the same GuHCl concentrations and various RNase A concentrations showed that the adsorption process of native RNase A is exothermic. That's because conformational loss occurred on adsorbed native protein and hydration associated with conformational loss (ΔΗ_mh) were predominant for the endothermic effect. The adsorbed amount is negative under this condition, so the water molecules were obviously combined with the protein molecules and packings on the surface, which was identical with the negative△H. The△H values of denatured RNase A by 0.3 mol/L GuHCl are all positive and decrease with increase of surface coverage, indicating that conformational loss and dehydration may be prior to adsorption affinity and hydration associated with conformational loss.