Studies on Barley Trypsin Inhibitor II. Structural Changes Induced by Denaturants and Their Reversibility

Publisher Website
Google Scholar
Abstract
No change in the activity of a trypsin inhibitor from barley was observed on treatment with heat and denaturants such as urea and guanidine hydrochloride. Thus, the conformational properties of the inhibitor were investigated. CD spectra of the native inhibitor were analysed by a curve-fitting technique using the data for poly-L-lysine. The results indicate that the inhibitor contains 41% α-helix, 25% β-structure, and 34% unordered conformation. In the presence of 2 emdash 8 M urea or 3 M guanidine hydrochloride, the CD spectra were almost unchanged. On treatment at 100° for 10 min in 8 M urea the depth of the trough at 200–240 nm decreased considerably, while both 6 M guanidine hydrochloride and 6 M guanidine hydrochloride at 100° reduced the molar ellipticity of the negative band at 222 nm by about two-thirds. When inhibitor treated in 6 M guanidine hydrochloride at 100° was diluted 10- or 30-fold, the conformation was significantly reformed. The CD spectra at alkaline pH showed that the inhibitor also has resistance to alkali. As a result of difference spectrum studies, it was shown that the inhibitor had peaks at 294 and 285 nm and troughs at 290 and 280 nm, due to denaturation, in strong denaturing media, 8 M urea at 100° and 6 M guanidine hydrochloride. The positive and negative peaks, however, immediately disappeared on removal of the denaturant. From spectro-photometric titrations, the phenolic hydroxyl groups were found to be ionized above pH 10.5. Four tyrosine residues are rapidly titrated and the last one was ionized above pH 12.5 with partial inactivation. Cleavage of the disulfide bridges in the inhibitor induced a very marked decrease in the value of [θ]232, leading to complete loss of the inhibitory activity. A possible interpretation of these data is that the inhibitor contains a rigid disulfide loop, and the disulfide bridges contribute to the structural stability and reversibility of the conformational changes of the protein.

This publication has 0 references indexed in Scilit: