On the Utility of Derivative and Multidimensional Fluorescence Spectroscopy for Studying the Intrinsic Fluorescence of Proteins

Abstract— The properties of hemocyanin from the terrestrial gastropod Achatina fulica, a protein with an exceptionally high molecular mass and unique structure, were studied using derivative and multidimensional fluorescence spectroscopy. Bovine serum albumin and rabbit muscle aldolase were chosen for comparison. The fourth derivatives of single total fluorescence spectra of the proteins proved to be the most informative. To compute the derivatives, an original script in the R open programming language was developed. The excitation wavelength was set to 280 nm in correspondence with the overlap of tyrosine and tryptophan absorption bands. The fourth derivatives were found to be indicative of individual features of proteins, and thus they may be regarded as their spectral “portraits”. In the future, this approach can be applied not only to characterize other proteins and investigate the influence of different factors on them, but also to study compounds of non-protein nature. Multidimensional fluorescence spectroscopy only confirmed the conclusions made when recording single spectra of intrinsic fluorescence of the proteins. With that, in general, it may help to define the optimal excitation and emission wavelength ranges of analytes and thus to refine the parameters of their handling.