Detection of Free Thiols and Fluorescence Response of Phycoerythrin Chromophore after Ultraviolet-B Radiation Stress

The chemistry of thiol-chromophore linkage plays a central role in the nature of fluorescence of phycoerythrin (PE). Interaction of thiol and chromophore is crucial for the energy transfer, redox signal and inhibition of oxidative damage. In the present investigation the effects of ultraviolet-B radiation on an emission fluorescence intensity and wavelength shift in PE due to interaction between thiol and chromophore by remarkable strategy of detection technique was studied. Purification of PE was done by using a gel permeation and ion exchange chromatography that yielded a quite high purity index (6.40) in a monomeric (αβ) form. UV-B radiation accelerated the quenching efficiency (24.9 ± 1.52%) by reducing fluorescence emission intensity of thiol linked chromophore after 240 min of UV-B exposure. However, after blocking of transiently released free thiol by N-ethylmaleimide, quenching efficiency was increased (36.8 ± 2.80%) with marked emission wavelength shift towards shorter wavelengths up to 562 nm as compared to 575 nm in control. Emission fluorescence of free thiol was at maximum after 240 min that was detected specifically by monobromobimane (mBrB) molecular probe. The association/dissociation of bilin chromophore was analyzed by SDS- and Native-PAGE that also indicated a complete reduction in emission fluorescence. Our work clearly shows an early detection of free thiols and relative interaction with chromophore after UV-B radiation which might play a significant role in structural and functional integrity of terminal PE.