Raman spectra of polypeptides containing L‐histidine residues and tautomerism of imidazole side chain

Abstract

SynopsisRaman spectra were measured for poly (L-histidine) in HzO, in D20, L-histidine in HzO, L-histidine-d3 (and d4) in DzO, and 4-methylimidazole in H20 with various pH (or pD) values. The Raman scattering peaks observed for these samples were ascribed to the neutral and positively charged imidazole groups on the basis of the spectral changes due to the pH variation and to the deuterium substitution of the imino protons. The vihrational modes of these peaks were deduced from the normal coordinate analysis made on the positively charged and neutral 4-ethylimidazoles. The Raman scattering peaks from the imidazole groups in the neutral form clearly indicate that these imidazole groups exist in the equilibrium between the two tautomeric forms, the 1-N protonated form (tautomer I) and the 3-N protonated one (tautomer 11). For example, the breathing vibration of the 1-N protonated form is observed a t 1282 cm-' for L-histidine and a t 1304 cm-' for 4-methylimidazole, while the breathing vibration of the 3-N protonated form is observed a t 1260 cm-' for I>-histidine and 4-methylimidazole. From the temperature dependence of the relative intensities of the tautomer I peak to that of the tautomer 11, it was concluded that the tautomer I is energetically more stable than the tautomer 11, and the AH value is 1.0 f 0.3 kcal/mol for L-histidine and 0.4 f 0.1 kcal/mol for 4-methylimidazole. Poly(L-histidine) with the neutral imidazole side chains shows the amide I peak at 1672 cm-', indicating that the sample assumes the antiparallel pleated-sheet structure. P o l y (~-A l a~~~-H i s~~) and p o l y (~-A l a "~~-H i s "~)were found to take the @-helical and P-form conformations, respectively.

Infrared and Raman spectroscopic study of pH-induced structural changes of l-histidine in aqueous environment

“…with either N p -or N t -protonated. Indeed, as empirically demonstrated by Ashikawa and Itoh [21,22] and confirmed by others [16,18,31], the 1588 cm À1 band appears to be correlated to the N p -protonated tautomer and the 1571 cm À1 one to the N t -protonated form. From the strong intensity of the latter compared to that of the 1588 cm À1 peak it can be concluded, that protonation of N t is preferred over protonation of N p , which is in line with the results of a temperature dependency studies of histidine by means of NMR [45] and of 4-methyl-imidazole by Raman [16].…”

“…The fact that both vibrations show only minor IR activity does not contradict to these assignments. It should be noted, however, that the empirical correlation of the 1285 cm À1 band to the N tprotonated and the 1265 cm À1 one to the N p -protonated tautomeric form of H 2 His 0 and HHis À as reported in the literature [16,21] is only valid for the pH range 9-13. As a shoulder at 1285 cm À1 is already present at pH 3, it implies that the bands are only useful as a marker for the conversion from one ionic state to another when the intensity is taken into account.…”

“…The band has been reported as an N-H in-plane bending [21] and as a ring stretching combined with =C-H bending [23,29] for histidine, and to a coupled ring dN-H/nC=N mode for 4-methyl-imidazole and 4-ethyl-imidazole [15,16,18]. Coupling of vibrations undoubtedly plays a role, and in agreement with [15,16,18,21] we believe the band to be predominantly an in-plane N-H bending/C=N stretching vibration for two reasons. Firstly, the band disappears upon deuteration, as shown by Toyoma et al [16], which implies that the ring nitrogens are largely involved.…”

“…On the other hand, it is obvious to assign the strong Raman band at 1269 cm À1 in acidic environment (pH <6) to an imidazole ring breathing vibration [21,22], regarding the high symmetry of the ring in the ionic forms H 4 His 2+ and H 3 His + . However, at pH >3, a second band around 1285 cm À1 grows in and both bands have been assigned to mainly a ring type of =C-H in-plane bending by Stewart and Fredericks [34].…”

“…For that reason, only results of theoretical calculations on the model compounds imidazole [12,13], 4-methyl-imidazole [12,[14][15][16][17] and 4-ethyl-imidazole [18] have been used to support the conclusions from empirical IR and Raman studies of histidine [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] and closely related molecules [37][38][39][40][41]. The majority of this research has been focussed on IR or Raman bands that can be used as a marker to determine the state of protonation of the imidazole nitrogen atoms [15,16,[20][21][22][23][24][25][26][27] or the type of metal ion binding [24,29,32,34,37,39,…”