Ultraviolet Difference Spectra and the Internal Structure of Proteins

(Received for publication, April 25, 1960) The absorption spectra of protein solutions in the region 250 to 310 rnp contain contributions from phenylalanine, tyrosine, and tryptophan residues. The band due to tyrosine is of particu- lar interest since it is found to be displaced by 1 to 6 rnp toward longer wave lengths in solutions of native proteins relative to free tyrosine or its peptides. This has been clearly demon- strated in denaturation, acidification, or hydrolysis .of proteins’ when the tyrosine band is usually shifted back to the blue.2 The extent of the shift is most conveniently and accurately measured in the form of a difference spectrum (2-4). The reasons for the shift have been the subject of considerable interest in recent years, since they may have an important bearing on the internal configurations of proteins. Spectral studies on insulin (4-6) and ribonuclease (7-9) have led to the suggestion that tyrosyl -OH groups are involved as donors in hydrogen bonds to other protein side chains. However, difference spectra very similar to those from proteins have been obtained with 0-methyltyrosine and other simple model compounds (10) where there is no possi- bility of hydrogen bonding in the above sense. In view of these and other data, it has been suggested (11) that protein difference spectra may arise indirectly from general configurational changes which lead to changes in polarity and polarizability in the vicinity of the tyrosyl groups. It, therefore, seems appropriate at this time to summarize the various effects which have been shown to give rise to ultraviolet spectral changes (on chromophores, in general) and attempt to determine which of these effects may be important in proteins. 1. The absorption band in the 250 to 310 rnp region can be displaced and altered in intensity for several reasons which may be grouped together as non-ionic solvent effects: a. Nonpolar solvents may produce displacements relative to the spectrum of the solute in the vapor phase (12-15). Polar solvents produce additional effects due presumably to their orientation around the solute chromophore. This orientation may stabilize dipolar excited states (12-15) and produce a red shift which increases with the dipole moment of the solvent. For the same reason, the band displacement to the red may increase with the polarizability or refractive index of the solvent (13, 16-