Near Ultraviolet Circular Dichroism and Absorption Spectra of Chicken Ovomucoid and Acetylated Derivatives at 297 and 77°K

Abstract

Abstract The spectral properties of tyrosyl side chains were investigated in chicken ovomucoid and derivatives produced by acetylating with N-acetylimidazole. Cooling ovomucoid to 77°K brings out the tyrosyl fine structure. Absorption spectra at 77°K reveal 2 different kinds of tyrosyl sites. About 4 tyrosyls have their longest wavelength absorption band at 284 nm and are relatively well shielded from the solvent. Approximately 2 tyrosyls, having their longest wavelength band near 282 nm, may be exposed to the solvent. These 2 tyrosyls are most easily O-acetylated. Among the 4 tyrosyls that are shielded from the solvent, 2 are especially difficult to acetylate. These 2 tyrosyls are O-acetylated only in the presence of 7 m guanidine hydrochloride. Completely acetylating all 6 tyrosyls in ovomucoid revealed that their molar absorptivity is about 20% greater than that of tyrosyls dissolved in water. If this hyperchromism is not taken into account, the number of acetylated tyrosyls calculated from spectral data will be inaccurate. Both acetylation experiments and 77°K spectra indicated that the near ultraviolet CD spectrum of native ovomucoid is contributed by tyrosyl, cystinyl, and phenylalanyl side chains. Positive tyrosyl CD bands at 284 and 277 nm are superimposed upon a broad, intense negative disulfide CD band. Acetylating the first 2 tyrosyls intensifies the tyrosyl CD fine structure by 25%. The tyrosyl CD bands disappear as the last 3 tyrosyls are acetylated and reappear upon deacetylating the O-acetyltyrosyls with hydroxylamine. The intense disulfide CD band with a maximum near 265 nm is not much affected by acetylating ovomucoid. Cooling the native protein to 77°K increases the disulfide rotatory strength about 30%. Reducing native ovomucoid with dithiothreitol abolishes both the disulfide and the tyrosyl CD bands.