Deep-ultraviolet Raman excitation profiles and vibronic scattering mechanisms of phenylalanine, tyrosine, and tryptophan

Abstract

Raman cross sections for excitation at wavelengths between 240 and 192 nm have been determined for resonance-enhanced ring modes of phenylalanine (Phe), tyrosine (Tyr), tyrosinate (Tyr"), and tryptophan (Trp) and for the breathing mode of S042" and the symmetric OH stretching mode of H20. The S042" and H20 bands, which are frequently used for relative intensity measurements, show strong preresonance enhancement at wavelengths below 220 nm, requiring large corrections to convert relative to absolute intensities. The aromatic amino acid intensity measurements were obtained with laser power levels in the linear regime and appear not to be influenced by saturation effects or photochemical transients. The 1210-cm"1 ring-C stretching mode of Phe and Tyr and the ring modes v12 (Phe, 1000 cm"1) or (Tyr, 850 cm'1) show strong enhancement in resonance with the allowed Bab transition and weak enhancement in resonance with the quasi-forbidden La transition. Their excitation profiles (EPs) peak on the red side of the La absorption band, consistent with destructive interference between La and Bab contributions to the /1-term Raman amplitude. The modes v8a, v8b (~1610, 1590 cm"1), and u9a (^-1180 cm"1), known to be responsible for the vibronic intensity of the La transition, are resonant with the La transition but also with the Ba b transition. Enhancement of v8b, which is antisymmetric in the molecular C2c symmetry, requires 5-term activity for the Ba b as well as the La states. The Ba b enhancement (192-nm excitation) of y8b, as well as v8a and v9a, increases in the order Phe < Tyr < phenolate « Tyr". This effect is suggested to arise from the substituent electronic perturbation, which leads to a Ba origin shift along v8a and i/9a and vibronic mixing between Ba and Bb via v8b. In the La region the EPs of v8a, vsb, and v9a are skewed to the blue side of the La absorption band, for Tyr more than Phe, suggesting constructive La-Bab interference for the 5 Raman terms. The Phe v8a and v8b overtones and combination bands are more strongly enhanced in resonance with Ba b than are the fundamentals; the 2v8a/v8a intensity ratio reaches a value of 2.1 at 192 nm. This effect is attributed to force constant changes in the allowed excited states. The 2v8a cross section is much lower for Tyr but increases again for Tyr", although the fundamental band is now much stronger. Evidently the Ba force constant change is diminished by the OH substituent, while the increased origin shift for O" increases the overtone intensity again. The Tyr 2u8b cross sections are about half those of Phe and the 2v8b/v8b ratio, ~0.15, is suggested to reflect the relative magnitude of C and 5 vibronic terms. Several Trp modes give EPs which track the strong 220-nm absorption band, attributed to Bb. These modes have significant fiveand six-membered ring involvement, implying delocalized character for the transition. The benzene-like v8a and v8b modes are enhanced in the short-wavelength absorption band (~1 95 nm), attributed to Ba. The »8a mode is also weakly resona...

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two‐state transition

“…Figure 8A shows selected DUVRR spectra of the soluble fraction of lysozyme samples. The major spectroscopic changes were evident for the C a -H bending mode and the 1000-cm À1 phenylalanine band (n 12 ring breathing vibrational mode [Matsuda et al 2003], in which alternating carbon atoms move toward and away from the center of the benzene ring) (Ziegler and Albrecht 1979;Asher et al 1986;Fodor et al 1989). The increase in the intensity of C a -H band indicated the melting of a-helix and the formation of an unordered structure.…”

Quantitative Evaluation of the Contribution of Weak Lysine-binding Sites Present within Apolipoprotein(a) Kringle IV Types 6–8 to Lipoprotein(a) Assembly

“…Protein concentrations for all r-apo(a) derivatives were obtained by absorbance measurements at 280 nm (corrected for Rayleigh scattering) using previously determined extinction coefficients (25). Protein integrity and purity were assessed by SDS-PAGE under non-reducing and reducing conditions followed by staining with Coomassie Blue.…”

“…Following gel filtration, proteins were dialyzed against 20 mM Tris-HCl pH 7.9, 1 mM EDTA for 2 h, and were then dialyzed against double-distilled H 2 O. Purified proteins were concentrated by lyophilization; before use, protein pellets were dissolved in 20 mM Tris-HCl, pH 7.9, and protein concentrations were determined by measurement of absorbance at 280 nm (corrected for Rayleigh scattering), using calculated extinction coefficients (E 0.1% ϭ 2.12 and 2.24 for KIV 7 and KIV 8 , respectively) (25). Protein integrity and purity were assessed by SDS-PAGE under nonreducing and reducing conditions, followed by staining with Coomassie Blue.…”

Sequences Within the Amino Terminus of ApoB100 Mediate Its Noncovalent Association With Apo(a)

“…Protein concentrations were determined by measurement of absorbance at 280 nm using corresponding molar extinction coefficients determined by the method of tyrosine difference spectroscopy. 26 Aliquots of the purified proteins were stored at Ϫ70°C before use.…”