Structure-function study on pepsin and comparative digestion of albumin and gliadins
Aresta-Dasilva, Stephanie K.
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Background: Although pepsin's crystal structure has been analyzed extensively, enzymatic components and sequential characteristics that make pepsin so functional and resilient in the acidity of the stomach, remain to be determined. Upon comprehending this phenomenon, researchers can begin to utilize and exploit such properties from other enzymes in an attempt to engineer enzymes that are gastric-active, with potential application in gastrointestinal (GI) disorders. Objective: The aim of this study was to investigate pepsin protease activities under ionic conditions representative of the stomach and representative of the duodenum. Two proteins were selected as protease substrates: albumin (test protein) and gliadin, which is an abundant dietary protein contained in cereals. The latter was chosen since it is a protein that is implicated in celiac disease. Celiac disease is a gastrointestinal (GI) disorder that renders patients intolerant to gluten and its molecular components, gliadins and gluten ins. Once dietary gluten reaches the duodenum of a celiac patient, T cells trigger an inflammatory response. Besides pepsin, we also investigated the susceptibility of both substrates to trypsin and chymotrypsin. All enzymes were standardized to the same unit concentration and digestion was carried out in solutions mimicking those of the stomach (pH 2.0 for pepsin) and the duodenum (pH 8.0, for trypsin and chymotrypsin). Enzymatic efficacy was assessed to determine which enzyme, at the standardized concentration, is most effective at digesting albumin and gliadins. Methods: Stimulated gastric fluid (SGF) was prepared, consisting of NaCl (35 mM) and HCl (84 mM). Albumin was dissolved to 2 mg/ml in water and gliadins in 60% ethanol stock solution to 2 mg/ml. Pepsin (3 ,900 U/mg solid) was dissolved in SGF to 1 mg/ml (3,900 U/ml). Chymotrypsin (66 U/mg solid) and trypsin (9 ,700 U/solid) were each dissolved to 1 mg/ml in duodenal buffer (DB). Gliadin or albumin were mixed with aliquots of pepsin, trypsin, or chymotrypsin yielding final albumin and gliadin concentrations of 100 µg/ml and final enzyme concentrations of 3.9 U/ml. The samples were incubated at 37 degrees in a waterbath. After various time points (t=O, 2, 5, 10, 20, and 30 min.), sample aliquots were removed and boiled to abolish pepsin enzymatic activity. Samples were then dried, re-suspended, and analyzed by SDS PAGE. After staining the gels with coomassie brilliant blue, proteolysis of albumin by pepsin was assessed visually and by densitometric analysis. Results: The SDS-PAGE gels revealed that the added amount of albumin was completely digested by pepsin after approximately 10 minutes of incubation. Mixed gliadins, however, remained mostly undigested throughout the entire incubatory period. Trypsin and chymotrypsin, however, at the tested unit concentration of 3.9 U/ml, were not effective at digesting albumin or gliadins throughout the entire incubatory period. Conclusion: Our results confirmed that gliadin is incompletely digested by pepsin in SGF solution. It is feasible that upon prolonged incubation of gliadin with pepsin, some degradation might occur, which will be further investigated. Furthermore, Trypsin and chymotrypsin are both ineffective when diluted to the same unit concentration as pepsin. Prolonging the incubatory time frame (t=0-30 minutes) may have some impact on digestion, however, it seems the most logical means of determining efficacy would be to determine the effective U/ml concentrations for trypsin and chymotrypsin. Only then can the efficacy of both duodenal enzymes towards albumin and gliadins be quantitatively compared to that of pepsin.
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