THERMAL PERTURBATION METHOD FOR ESTIMATION OF EXPOSED TYROSINES OF PROTEINS .I. RIBONUCLEASE IN AQUEOUS GLYCOL, GLYCEROL, AND DENATURANTS

A new spectral method is presented for the estimation of exposed chromophores of proteins, and has been applied to the tyrosines of bovine pancreatic ribonuclease. The method is that of thermal perturbation, and is based on the change in spectrum arising from the change in solvation of the chromophore accompanying a change in temperature. Difference spectra are taken from identical solutions at two temperatures, one usually 25°, the other usually 4°, but the temperature range used must be outside the range of a thermal conformational transition. For the tyrosine chromophore the difference absorption band at around 290 mμ is used, and ∆ε290 for RNase is divided by ∆ε290 for the model compound to give the number of exposed tyrosines. For ribonuclease in the denaturants guanidinium chloride and LiBr, the numbers of exposed tyrosines are in good agreement with estimates made by other methods. For ribonuclease in H2O 3.6 exposed tyrosines were found, in agreement with other spectral data and with X-ray data. For ribonuclease in 75-97% glycerol or ethylene glycol the number of exposed tyrosines is greater than indicated by other methods. © 1969, American Chemical Society. All rights reserved.