Tyrosine Hydrogen-Bonding and Environmental Effects in Proteins Probed by Ultraviolet Resonance Raman Spectroscopy

Ultraviolet resonance Raman spectra with 229-nm excitation are reported for aqueous tyrosine and for ovomucoid third domain proteins from chicken [OMCHI3(-)] and from chachalaca [OMCHA(-)], as well as α₁, β₂-, and β-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes ν_8aand ν_8b. The v_8bfrequency decreases with the degree of Tyr H-bond donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound β-cresol in H-bond acceptor solutions for which the enthalpy of H-bond formation can be obtained from the literature. With this calibration it is possible to estimate Tyr H-bond enthalpies in proteins for which Tyr is a H-bond donor; values of 13.7, 9.6, and 11.2 kcal/mol were found for OMCHA3(-) and for α₁- (or a₂-) and β-purothionin, respectively. The intensity of the 1176-cm^-1ν_9aband of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm^-1Fermi doublet excited at 200 nm both correlate strongly with the estimated H-bond enthalpies, but large deviations are seen for the purothionins, reflecting a special environment for the Tyr residue of these proteins, which is believed to be constrained in a hydrophobic pocket. The molar intensity of the strong ∼1000-cm^-1v₁₂band of phenylalanine in aqueous solution is about half the value observed in most proteins. Addition of ethylene glycol to aqueous phenylalanine increases the intensity, which attains a value similar to those seen in proteins. Protein environmental effects on UVRR intensities for aromatics are expected to be common.