Spectroscopic and calorimetric studies of congo red dye-amyloid peptide complexes

Thermodynamic properties of complexes of Congo Red (CR) dye with amyloid  (A) peptides were studied by both absorption spectroscopy and isothermal titration calorimetry (ITC). Based on the absorption spectrum for the formation of CR-Aβ complexes in phosphate buffered saline solution (pH 7.4), van’t Hoff plots over a temperature range of 10oC to 70oC were created for CR-Aβ1-40, -Aβ12-28, and -Aβ1-42. The plot for CRAβ12-28 complex showed a relatively linear feature within the given temperature range with H = –10.1 0.6 kJ/mol and S = + 0.128  0.002 kJ/(mol K). However, the plot for CR-Aβ1-40 and CR-Aβ1-42 complexes exhibited two distinct linear regions with opposite slopes centered at a specific temperature, Ts, which was 54.7  0.2°C and 34.8  0.2°C, respectively. The ITC experiments conducted at 25°Cin water exhibited quite a different situation from the above mentioned spectroscopic approach. The ITC studies yielded a H of –85.3  0.2 kJ/mol for the CR-Aβ12-28 complex with negative entropy change –0.152 kJ/mol K). For CR-Aβ1-40, the ITC studies indicated the presence of two binding sites with H1 = –81.8  0.3 kJ/mol and H2 = –119.5  0.2 kJ/mol with K1 = 5.5  0.7  10 M and K2 = 6.9  2.4  10 M, respectively. These binding constants are consistent with the model suggested by several studies. Both binding sites showed negative entropy changes suggesting that the formation of the complex is enthalpically driven. The disagreement in thermochemical values between two different methods confirmed that the enthalpy and entropy are heavily dependent on temperature and buffer/salt environment, and may involve inherently different reaction paths.