The study of structure and stability of Carboxypeptidase A in the presence of Spermine by spectroscopy methods

Carboxypeptidase A (CPA) (EC 3.4.17.1) is a zinc-containing proteolytic enzyme which removes the C-terminal amino acid from a peptide chain with the free carboxylate-terminal. Polyamines such as spermine are low-molecular-weight aliphatic polyamines, that are involved in the biology of protein aggregation. In this study, the effect of spermine on the structure and thermal stability of CPA was investigated by ultraviolet−visible (UV−vis) spectroscopy, intrinsic fluorescence and steady-state thermal stability techniques at the pH of 7.5. Forming a complex between CPA and spermine caused the decrease in the absorbance intensity of the CPA. In addition, the thermal stability (Tm) of CPA in the presence of spermine was enhanced with increasing the concentration of spermine. Spectrofluorometric results proved that with the addition of spermine to the protein solution, the emission intensity of CPA was extremely reduced. The results of fluorescence intensity changes, at two temperatures of 308 and 318 K, also suggested that spermine had a great ability to quench the intrinsic fluorescence of CPA through the quenching procedure. The thermodynamic parameters such as ΔH and ΔS were calculated to be negative, indicating that the interactions between spermine and CPA were mainly due to van der Waals forces or hydrogen bonding. The results also showed that binding of spermine to CPA could cause conformational changes in CPA.

Keywords:

Spermine , Carboxypeptidase A , UV-Vis spectroscopy , Fluorescence spectroscopy , Thermal stability