Studies on Barley Trypsin Inhibitor
II. Structural Changes Induced by Denaturants and Their Reversibility
1976 Volume 79 Issue 2 Pages 321-328
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1976 Volume 79 Issue 2 Pages 321-328
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 2-8M urea or 3M guanidine hydrochloride, the CD spectra were almost unchanged. On treatment at 100° for 10min in 8M urea the depth of the trough at 200-240nm decreased considerably, while both 6M guanidine hydrochloride and 6M guanidine hydrochloride at 100° reduced the molar ellipticity of the negative band at 222nm by about two-thirds. When inhibitor treated in 6M 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 285nm and troughs at 290 and 280nm, due to denaturation, in strong denaturing media, 8M urea at 100°C and 6M guanidine hydrochloride. The positive and negative peaks, however, immediately disappeared on removal of the denaturant. From spectrophotometric 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]222, 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.
