Ultraviolet difference spectra

D. A. Chignell and W. B. Gratzer, Solvent effects on aromatic chromophores and their relation to ultraviolet difference spectra of proteins, J. Phys. Chem. 72, 2934-2941 (1968). 

Ultraviolet difference spectra have frequently been used to measure the ionization of the phenolic hydroxyl of tyrosines. The sulfydryls of cysteines and the imidazoles of histidines are also amenable to difference spectroscopy. 

The secondary and tertiary structure of a partially purified 7S globulin was examined by Fukushima (7) based on optical rotatory dispersion, infrared and ultraviolet difference spectra. Antiparallel (5 -structure (352) and random coil (60%) predominated with only 5% helical structure present. The contribution of the three structures was calculated from molecular ellipticity values obtained by circular dichroism (11) and from the Moffitt parameters in ORD (11, 12). Between 210 and 250 nm, the experimental CD curve for the 7S protein was similar to the CD curve computed from ORD Moffitt parameters with the major dissimilarity occurring at 208-213 nm. 

Relative to secondary structure, viscosity, sedimentation velocity, ultraviolet difference spectra and optical rotatory dispersion studies (4,24,25) showed that glutenin appears to be an assymetric molecule with a low a-helix content (10-15%). Glutenin contained more a-helix structure in hydrochloric acid solutions and less in urea solutions. The amount of a-helix structure is also influenced by changes in ionic strength (26). 

Eisenberg, D. S. (1961). Ultraviolet Difference Spectra of Human Serum Albumin and Reduced-Carboxymethylated Albumin. B. A. Honors Thesis, Harvard University, Cambridge, Massachusetts. 

Chart I (Ramachandran and Witkop, 1959) summarizes these results The two sequences Try-Ala (17-18) and Try-Lys (52-53) are exposed and fully reactive NBS the sequence Try-Thr (151-152) is buried (cf. Herskovits and Laskowski, 1960) in the A-protein, but becomes accessible to NBS in the tryptic hexadecapeptide. In addition to the methods of spectral perturbation of chromophoric groups by polyhydroxy compounds, spectrophotometric titration studies, and ultraviolet difference spectra the reactivity of tryptophan toward selective agents such as NBS may offer a new tool for assessing influences of secondary or tertiary structure in this area. 

In summary, these experiments illustrate the use of titration curves and ultraviolet difference spectra to obtain information about interactions which render some of the carboxyl groups of lysozyme abnormal. 

Bigelow (1960) had previously interpreted his ultraviolet difference spectra measurements in terms of hydrophobic bonding, and had indicated that a tyrosyl-carboxyl hydrogen bond probably does not exist. However, Bigelow s measurements were made at 25°C. therefore, he could not detect the effect of the hydrogen bond since the latter manifests itself most clearly at low temperature (transformation II to III of Fig. 167). 

Liudskog, S., and H. Nilsson Location of Tryptophanyl Groups in Human and Bovine Carbonic Anhydrases. Ultraviolet Difference Spectra and Chemical Modification. Biochim. Biophys. Acta 295, 117-130 (1973). 

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