UV-Light Exposure of Insulin: Pharmaceutical Implications upon Covalent Insulin Dityrosine Dimerization and Disulphide Bond Photolysis
Figure 8
Concentration of detected free thiol groups (open circles) in human insulin vs 276 nm exc. time.
Detection of free thiol groups was carried out using the Ellman’s assay before and after 276 nm light continuous exc. (0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, and 2.5 h) of human insulin in solution. The concentration of free thiol groups was estimated from the absorbance of the Ellman’s assay reaction product, TNB2−, at 412 nm (ε412 nm = 14150 M−1.cm−1 [44]). The experimental values were fitted using an exponential function y = y0– A.e−R0t (fitted curve in red), where y is the concentration of thiol groups (µM) at the 276 nm excitation time t (h), y0 and A are constants and R0 is the rate of thiol group formation (h−1). Fitted experimental parameters were: y0 = 5.04±0.24 µM, A = 5.27±0.22 µM, R0 = 0.87±0.09 h−1. Root mean square error was 99.41%.