Second derivative analysis of synthesized spectra for resolution and identification of overlapped absorption bands of amino acid residues in proteins: Bromelain and ficin spectra in the 240-320 nm range.

Abstract

We establish the origin and formation of peaks in UV absorption spectra of proteins by applying the second derivative analysis to (i) spectra of the native protein, (ii) to its model spectra "synthesized" as a sum of partial free amino acid spectra and (iii) to absorption spectra of the free amino acids. We show that the bromelain peaks at 248.2, 253.2, 258.4 and 264.2 nm are due to phenylalanine maxima; the predictable peak at 279.6 nm (which is almost coincident with the extremum of the zero-order spectrum at 279.4 nm) is mainly due to tyrosine maximum, while the peaks at 274.6 and 290.6 nm are due to tryptophan maximum; 268.0 nm peak to the superposition of tyrosine and phenylalanine maxima, and 283.4 nm peak to the superposition of tyrosine and tryptophan maxima. Similar results are obtained for ficin: the peaks at 248.4, 253.0 and 258.8 nm are formed by the phenylalanine maxima, the predictable peak at 264.4 nm accords with the corresponding bromelain 264.2 nm peak; the 279.4 nm peak almost coincides with the zero order spectrum peak (279.6 nm), but it is expressed stronger than that of bromelain due to a different ratio of tyrosine to tryptophan side groups. The peaks at 273.4 and 290.6 nm are associated with tryptophan, the 268.0 nm peak being mainly due to tyrosine (and fractionally to phenylalanine); and the 283.8 nm peak belongs to tyrosine and, to a greater extent, to tryptophan. We demonstrate that the amino acid residues of tryptophan, tyrosine and phenylalanine undergo correspondingly the largest, intermediate and the lowest positive (red) wavelength shift in the zero-order protein absorption spectrum with respect to the model (synthesized) spectrum. The difference appearing in the positions of the bromelain and ficin absorption band peaks is determined by superposition of relative contributions from amino acid residues. This superposition is resulted from (i) linear combination of amino acid residues spectra and (ii) their different (non-uniform) wavelength shifts as functions of microenvironment of these residues' chromophores. The proposed approach to the analysis of the protein absorption spectra with the help of "synthesized" spectra can be transferred to other objects studied in analytical and organic chemistry of high molecular compounds containing monomer units with various chromophores.