Circular Dichroism (CD)

The CD measurements were recorded with a CD spectrophotometer (Chirascan, Applied Photophysics). Far-UV (200–260 nm) CD was used to investigate the secondary structural changes in insulin with a cuvette cell of path length 0.1 cm. Spectra were recorded for free insulin and Zn–insulin in the presence and absence of protamine. Phenol was added to Zn–insulin in a 3:1 molar ratio to maintain a hexameric state. The concentrations of insulin and ZnSO₄ were 20 μM (0.116 mg mL^–1) and 10 μM (0.0017 mg mL^–1), respectively. All of the experiments were performed in doublets. Changes observed in the helical content were calculated using online software BeStSel (β Structure Selection), as shown in Figure ​Figure22C.

Far-UV CD of (A) free insulin and Zn–insulin and that of (B) insulin–protamine, Zn–insulin–protamine, and protamine. The concentrations of protamine, insulin, and ZnSO₄ were 20, 20, and 10 μM, respectively. (C) % Helical content of insulin and Zn–insulin with and without protamine. Thermal stability curve of (D) free insulin and Zn–insulin and (E) insulin–protamine and Zn–insulin–protamine using ultraviolet–visible (UV–vis) spectrometer. (F) Plot of CD ellipticity at 208 nm vs temperature curve of insulin–protamine and Zn–insulin–protamine. Table showing a comparison of T_m values. Far-UV CD melting spectra of (G) insulin–protamine and (H) Zn–insulin–protamine. Phosphate buffer (10 mM) containing 100 mM NaCl at pH 8.0 was used. The insulin/Zn molar ratio was 1:0.5, and the insulin/phenol molar ratio was 1:3.