Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy

Abstract

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 .alpha.1-, .alpha.2-, and .beta.-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes .nu.8a and .nu.8b. The .nu.8b frequency decreases with the degree of Tyr H-bound donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound p-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 .alpha.1- (or .alpha.2-) and .beta.-purothionin, respectively. The intensity of the 1176-cm-1 .nu.9a band of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm-1 Fermi 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 .apprx. 1000-cm-1 .nu.12 band 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.