Investigation by Synchrotron Radiation Circular Dichroism of the Secondary Structure Evolution of Pepsin under Oxidative Environment

Abstract

Food processing affects the structure and chemical state of proteins. In particular, protein oxidation occurs and may impair protein properties. These chemical reactions initiated during processing can develop during digestion. Indeed, the physicochemical conditions of the stomach (oxygen pressure, low pH) favor oxidation. In that respect, digestive proteases may be affected as well. Yet, very little is known about the link between endogenous oxidation of digestive enzymes, their potential denaturation, and, therefore, food protein digestibility. Thus, the objective of this study is to understand how oxidative chemical processes will impact the pepsin secondary structure and its hydrolytic activity. The folding and unfolding kinetics of pepsin under oxidative conditions was determined using Synchrotron Radiation Circular Dichroism. SRCD gave us the possibility to monitor the rapid kinetics of protein folding and unfolding in real-time, giving highly resolved spectral data. The proteolytic activity of control and oxidized pepsin was investigated by MALDI-TOF mass spectrometry on a meat protein model, the creatine kinase. MALDI-TOF MS allowed a rapid evaluation of the proteolytic activity through peptide fingerprint. This study opens up new perspectives by shifting the digestion paradigm taking into account the gastric digestive enzyme and its substrate.

Keywords: digestion; mass spectrometry; pepsin; protein oxidation; synchrotron radiation circular dichroism.

Figure 1

SRCD spectra of pepsin (A) and oxidized pepsin at 3 mM FeSO4/DETAPAC/H2O2 (B). The kinetic of gastric acidification was monitored, from 0 to 120 min. Spectra were recorded every 30 min and colored from dark blue, light blue, pink, orange, and dark red, respectively. For clearness, error contours at 1 sigma were drawn only for the first and last spectra, given their similarity for the other spectra. (C) Evolution of the percentage in α-helix in control (in red) and oxidized (in blue) pepsin during the kinetic of gastric acidification, from 0 to 120 min.

Figure 2

MALDI-TOF spectra of creatine kinase digestate by pepsin (A) and oxidized pepsin at 3 mM FeSO₄/DETAPAC/H₂O₂ (B) monitored through the kinetic of gastric acidification, from 0 (in red) to 120 min (in dark green), to pH 5 to pH 2, respectively. MALDI-TOF individual spectra (n = 9 per each condition) are represented as a gel-view in the upper part. Principal component analysis of peptide spectra consists of the score plot on the upper part and the loading plot on the lower part.