The thermodynamical stability and remained activity of mushroom tyrosinase (MT) fromAgaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40°C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obeye the first order law. Inactivation rate constant (k_inact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of k_inact(0.7×10⁻⁴s⁻¹) in comparison with their absence (2.5×10⁻⁴s⁻¹). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodyanamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.

The effects of DPG, IHP, GTP, GDP and GMP on the structure and stability of haemoglobin were electrochemically investigated with an iodide-modified silver electrode in 0.01 M KNO3 at pH 7.0. Anodic and cathodic peaks of haemoglobin were observed at 250 mV and 12 mV with a formal potential value of 133 mV vs. Ag/AgCl. The effects of different concentrations of DPG, IHP, GTP, GDP and GMP on the anaerobic redox reaction were determined. The results showed that DPG and IHP can lead to a positive shift in the reduction peak of haemoglobin, indicating that the oxidation peak shift of haemoglobin was small as a result of stabilization of the reduced state and destabilization of the R-like state of haemoglobin. GTP elicited a more positive shift in the cathodic and anodic peaks of haemoglobin at a higher concentration, signifying that it has a low-affinity binding site on haemoglobin. The positive shift of the cathodic and anodic peaks revealed a slight variation in the structure and indicated the unfolding of haemoglobin in the presence of high concentrations of GTP. Our study also showed that GDP and GMP did not cause significant shift the cathodic and anodic peaks of haemoglobin even at high concentrations, refuting the existence of specific GDP- and GMP-binding sites on the protein. Moreover, the iodide-modified silver electrode method proved to be easy and useful in investigating the effects of ligands or other effectors on haemoglobin in solution.