This paper relates to the further characterization of a novel peptide-bond-derived blue-green visible fluorescence first described by us. In this paper, we show that the excitation maximum is at ~387 nm in concentrated solutions of the protein, gamma-B crystallin. There is no excitation below 300 nm, demonstrating that the fluorescence does not owe to aromatics, or their immediate photoproducts (such as N-formylkynurenine). The emission maximum, in this case, turns out to be ~360 nm; however, we have already shown that this emission maximum wavelength is protein dependent, and also dependent on the relative contents of different secondary structural elements. We use time-resolved fluorescence studies to show that there are three components to the emission at 360 nm, two of which have lifetimes in the nanoseconds range; the third is sub-nanosecond. With lysozyme, we show that the fluorescence is altered with denaturation. We also show that the fluorescence is altered with heightened protein concentration, with gamma-B crystallin, owing to conformational changes in this protein which are effected by changes in protein concentration. Using very high protein concentrations, we also show that the excitation transition of this peptide-bond-derived fluorescence is associated with differential absorption of right- and left-circularly polarized light, i.e., with an interesting CD spectrum. Thus, there is also a previously-unnoticed, long-wavelength CD band which is associated with peptide bonds in the region of the boundary of the UV and visible ranges of electromagnetic radiation.

Click to Read in Detail