| Popis: |
Pepsin is an aspartic acid protease and the first protease that proteins encounter in the gastrointestinal tract. In vitro digestion assays on proteins including those found in food, or as oral protein and peptide drugs, often involve different solvents or pH conditions where the activity of the digestive enzyme may not be optimal. To improve the accuracy of these assays it is, therefore, important to understand the effect of common experimental conditions, such as solvent, on enzymes such as pepsin. Herein we investigate the activity and structure of porcine pepsin in H2O and D2O at pH values between 1–8. When dissolved in D2O, the enzyme activity of pepsin between pH 1–3 decreased to 60% of the activity in H2O. However, the relative change in activity with pH was similar in D2O and H2O. CD measurements demonstrated that changing the pH and solvent did not influence the secondary structure of the pepsin enzyme. SAXS analysis revealed that structural changes to pepsin did not occur until a pH value between pH 7 and 8, at which point it was denatured and adopted an extended state. Therefore, changes in the pepsin enzymatic activity with pH and solvent change were found to be related to the solubility of pepsin but not to the structure of the protein. In digestion studies, pepsin activity is determined based on its measured activity in H2O at pH 3, regardless of the dynamic pH conditions in digestion or the local environment of the pepsin. This research has therefore significant applicability in improved setups for future digestion and drug bioavailability experiments, as well as future neutron scattering, NMR, and FTIR experiments for enzyme studies in D2O. |
