Difference Spectrum and Enzymatic Activity of Denatured Chymotrypsin

Abstract

WHEN chymotrypsin is heat denatured in dilute solution at pH of 3.0 in 0.001 M hydrochloric acid, or at the same pH in 0.01 M citrate, changes occur in the ultra-violet absorbance spectrum. When the differences in absorbance between the native and denatured enzyme are determined (difference spectrum) peaks are obtained at 231.5, 285.5 and 293 mµ (Fig. 1). These peaks have been found in a wide variety of proteins when denatured by various means^1,2. The peaks in the vicinity of 280–290 mµ have been attributed to changes in the environment of the chromophores, tryptophan and tyrosine brought about by conformational changes occurring in the denaturation process. The spectral differences occurring around 230 mµ, while influenced by these amino-acid residues, have been attributed to structural alterations other than those associated with tryptophan and tyrosine².