title: Ethanol and acetonitrile induces conformational changes in porcine pepsin at alkaline denatured state.
abstract: Pepsin, a member of the aspartate protease family, exists in a partially unfolded state at alkaline pH where the N-terminal domain of pepsin has a flexible structure while the C-terminal domain has a highly folded structure. In this work, the conformational stability of porcine pepsin in an alkaline denatured (A(D)) state against acetonitrile and ethanol solvents was studied using a combination of electronic circular dichroism (ECD) and fluorescence techniques. The ECD results demonstrate that both ethanol and acetonitrile induce secondary structural changes in pepsin at A(D) state. However, the minimum concentration required to induce significant secondary structural changes in pepsin varies for ethanol (>30%, v/v) and acetonitrile (>60%, v/v) solvents. At maximum concentration used (90%, v/v), both solvents induce predominantly β-sheet conformation. Unlike acetonitrile, ethanol induces significant amount of non-native α-helical conformations at the intermediate concentrations (50-80%). The tryptophan fluorescence results demonstrate that both acetonitrile and ethanol induce substantial changes in the tertiary structure of pepsin in the A(D) state above certain concentrations. The current results have important implications in understanding the effect of co-solvents on the conformation of proteins in the "denatured state".
authors: Bioorganic Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India. ganesh@clri.res.in
authors: Ganesh
authors: G
authors: Shanmugam
authors: C Chinnarul
authors: CC
authors: Selvi
authors: Asit Baran
authors: AB
authors: Mandal
mesh_terms: D000097:Acetonitriles / Q000494:pharmacology*; D000818:Animals; D000431:Ethanol / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D010434:Pepsin A / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects*; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013552:Swine
chemical_list: D000097:Acetonitriles; D000431:Ethanol; D010434:Pepsin A; C032159:acetonitrile