Abstract:

The effect of solution composition on the thermostability of aconitase conformation has been studied by differential scanning calorimetry. In order to know the change of the active site of aconitase ,the three salt links in the active site in the pairs Asp 100•His 101,Asp 165•His 147,and G1u 262•His 167 have been studied by increasing temperature and pH of the solution. At pH 7.47,the calorimetric results demonstrated that the three salt links were broken during heat denaturation and followed by deprotonation of the cationic imidazole groups. Consequently, the number of protons bound to the enzyme molecules in native state is higher than that in denatured state. When pH value was raised to 7.89 or higher, the three salt links could not be formed, consequently Tp/pH was equal to zero. The thermodynamic calculation suggested that the fourth or fifth paired residues as Asp 100•His 101 might exist within the aconitase molecules. The experiments showed that the salts NaCl and NaSCN can raise the thermostability of aconitase, but NaSCN is more effective under the same ionic strength. 0.2 mol•L-1 of NaSCN has the highest ability to raise the thermostability. The chelants SA and OP with antitumor activity can not eliminate the Fe-S cluster within the aconitase molecules and almost not influence its thermostability, but considerably change its denaturational enthalpy. The latter suggests that some relationships may exist between the thermodynamic parameter of enzyme denaturation and antitumor activity of the chelants.

Key words: Aconitase, Salt link, Thermostability, Differential scanning calorimetry