Solvent effect on The conformational Modification and in vitro animal skin permeation of Lysozyme model Peptide

Abstract

The present study was aimed to characterize lysozyme in its molten globule (MG) or partially folded state and to study the in vitro animal skin penetration. The characteristics of MG state are represented as a loss in the tertiary structure, while retaining the native-like secondary structure and an evidence of compactness. This study was done to determine the effects of aqueous-ethanolic environment, acidic environment and the combined environment on the conformational modification of lysozyme. The evidence of MG state occurred in the conditions which composed of lysozyme in 80% v/v of ethanol in the aqueous-ethanolic solution [L(80)(0)] or 35% v/v or 40% v/v of ethanol in the aqueous-ethanolic solution in 20 mM of HCl acid [L(35)(20)) or L(40)(20), respectively]. Moreover, the modified lysozyme in above conditions was shown to be efficiently bound to 1-anilino-8-naphthalene sulfonate anion (ANS) which signified MG state. This state could be reversed to native conformation when diluted with water at twenty times its initial volume. Nevertheless, the acidic environment alone could not induce lysozyme to MG state. Lysozyme dissolved in 4 N HCl overnight led to protein aggregation while the structure of lysozyme was completely destroyed in 6 N HCl. The solutions of L(80)(0) and L(35)(20) were selected as solvents to induce lysozyme to MG state for the penetration experiment using pig’s ear skin as an in vitro model skin. The effects of vehicles on the integrity of the skin were determined using hydrophilic propranolol HCl as a marker. Although these vehicles did not cause leakage of model skin, they decreased the penetration of propranolol HCl. Determination of the skin surface after ethanol exposure using Cryo-SEM showed dehydration and lipid extraction of the skin. Exposure of the skin to the combined environment, where both aqueous-ethanol and acid were presented, demonstrated swelling and changes in the skin structure on the model skin. The determination of the amount of lysozyme remaining in the donor compartment showed that the lysozyme from L(80)(0) and L(35)(20) disappeared from the donor side faster than that from L(0)(0) and L(35)(0). The reason for this finding still needed further investigation. However, penetration of lysozyme in MG state with increasing hydrophobic character was one of the possibilities.