Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology

Continuous 295nm excitation of bovine apo-α-lactalbumin (apo-bLA) results in an increase of tryptophan fluorescence quantum yield and in a progressive red-shift in tryptophan fluorescence emission. Furthermore, 295nm excitation in apo-bLA leads to disulphide bridges breakage, leading to free thiol groups. Thiol detection has been confirmed with Ellman's reaction. The increase in thiol concentration with illumination time is correlated with the increase in fluorescence quantum yield. Once broken, disulphide bridges no longer efficiently quench protein fluorescence. After 2h of continuous excitation a putative isosbestic point is observed in tryptophan fluorescence emission spectra, suggesting equilibrium between two pools of molecules: one with intact disulphide bridges and low fluorescence quantum yield, and another pool with disrupted disulphide bridges, high fluorescence quantum yield and red-shifted emission. Normalized excitation spectra show that 295nm excitation induces spectral changes in the absorption spectrum of tryptophan. The rate of Trp fluorescence quantum yield increase with excitation time increases with temperature between 9.3^oC and 29.9^oC. Experimental Ahrrenius activation energy was 21.8 ± 2.26 kJ.mol^-1. Photoionization mechanism(s) of Trp in proteins and in solution and the activation energy of Trp photoionization leading to disulphide bridge disruption in proteins are discussed and correlated. The observations reported in the present paper rise some doubt on the conclusions made in many fluorescence spectroscopy studies of proteins. Prolonged illumination of any disulphide bridge containing protein will have photochemical consequences, such as disulphide bond disruption. We may not be able to observe proteins using UV light without perturbing them.