Прецизионная интерферометрия как новый метод исследования конформационного состояния белка и его взаимодействия с растворителем

Abstract

A new method for studying the conformational state of a protein and its interaction with a solvent has been developed. The method is based on measuring the refractive index of a protein solution using a precision laser interferometer with an accuracy of 1 • 10–7. The interferometer was calibrated based on the known temperature dependence of water refractive index and concentration dependence of the refractive index of a NaCl solution. Using an interferometer, changes in the refractive index during proteolysis (an increase of 9 ∙ 10-6 and 2.4 ∙ 10-6 in the refractive index of bovine serum albumin and egg lysozyme solutions during pepsin-catalyzed hydrolysis) and denaturation (increase in the refractive index by 4.5 ∙ 10-5 during the reaction of egg lysozyme with guanidine hydrochloride and dithiothreitol) have been first recorded. Based on direct interferometric measurements, the widespread assumption has been refuted that the refractive index of protein solutions is determined only by the concentration of the protein and its amino acid composition and does not depend on the state of protein fragmentation. The increased accuracy of the interferometer allows one to study the increment of the refractive index (dn/dc) at low concentrations of dissolved compounds (<< 1%), as well as processes leading to a change in the conformation of macromolecules in solution.