Synopsis
The pH, temperature, and reagent concentration dependences of the cyanuration of tyrosines within proteins are compared with those of the free amino acid. The results indicate that the mechanism of the cyanuration reaction is a nucleophilic aromatic substitution of the S N ~ type, where the tyrosine oxygen acts as a nucleophile and the displacement takes place at a carbon atom which is part of the six-membered ring of the striazene. This reaction must compete with the hydrolysis of cyanuric fluoride in aqueous media.\ These competing reactions generate curves for the pH dependence of the reactivity of tyrosine with cyanuric fluoride which vary in a predictable manner with the extent of ionization of tyrosine and the concentration of cyanuric fluoride. In the case of proteins, the dependence of the degree of tyrosine reactivity on pH and temperature within the pH range of full reactivity of the free amino acid reflects constraints imposed by the microenvironment of these groups within the three-dimensional structure of the macromolecule.
Cyanuric fluoride (CyF) is one of the commonly used tyrosine residue modifying reagents. p 2 z 3 This modification leads to 0-derivatives. The substitution of a highly electron-withdrawing group on the oxygen affects the T-T* transition which is responsible for the 275-nm absorption band. This results in the diminution of the intensity of UV absorption and a displacement of the 275-nm band to a lower wavelength. Similar effects are shown by 0-acetyl tyrosine4 and tyrosine-O-sulfate.5 In the case of proteins, this reaction is carried out only within the pH limits of 9.3 to 12..5. Furthermore, the selection of pH's should be such that no time-dependent conformational changes occur in the protein.6 This is essential if one is to avoid ambiguous results and to obtain information on group exposure in the native or nearly native state.
Within these limits of pH, the extent of tyrosine residue cyanuration in proteins is a strong function of pH6-* and, with a few exception^,^*'^ of temperature. The difference in the reactivity of individual tyrosine residues incorporated in various regions of the three-dimensional framework of protein molecules has been attributed to constraints imposed on the residues by their immediate environment, which reflect the secondary and * Publication No. 859.