Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable

Abstract

The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.

Fig 1. pH effect on pepsin degradation of three proteins (HRP, Rubisco, Hb) at 10 U:1 μg ratio for 2 minutes.

Panel A: The amount of Coomassie Blue stained intact protein after exposure to pepsin for 2 minutes at each condition was quantified and is shown as a percentage relative to the amount of starting material. For Rubisco, only the large subunit (LS) was quantified as described in Results section. Panel B: Three gels from triplicate pepsin degradation assays with HRP. The gel lanes are: 1: MW, 2: Pepsin Only, 3: HRP protein Only, 4: 0 minute, Pepsin + HRP protein, 5–12: HRP exposed to pepsin for 2 min at pH 1.2, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, and 6.0, respectively.

Fig 2. Effect of pepsin-to-substrate protein ratio and time on pepsin degradation of Rubisco LS at six different pHs.

The amount of Coomassie Blue stained intact protein at each condition was quantified and is shown as a percentage relative to the amount of starting material. A) 10 U:1 μg; B) 1 U:1 μg; C) 0.1 U:1 μg.

Fig 3. SDS-PAGE analysis of pepsin degradation of HRP, Rubisco, and Hb at pH 1.2 and three pepsin-to-substrate protein ratios.

1 μg of substrate protein, based upon the pre-degradation concentration, was loaded in each well. For all figure panels, the gel lanes are: 1: MW, 2: Pepsin Only, 3: Substrate Protein Only, 4: 0 minute, Pepsin + Substrate Protein, 5–11: 0.5, 2, 5, 10, 20, 30, 60 minute(s), respectively, of Pepsin + Substrate Protein. Figure Panels: A) Rubisco, 10 U:1 μg; B) Rubisco, 1 U:1 μg; C) Rubisco, 0.1 U:1 μg; D) HRP, 10 U:1 μg; E) HRP, 1 U:1 μg; F) HRP, 0.1 U:1 μg; G) Hb, 10 U:1 μg; H) Hb, 1 U:1 μg; I) Hb, 0.1 U:1 μg.

Fig 4. Quantification of degradation of HRP, Rubisco LS, Hb, STI and LTP at various pH conditions and pepsin-to-substrate protein ratios after 60-minute incubation with pepsin.

The amount of Coomassie Blue stained intact protein at each condition was quantified and is shown as a percentage relative to the amount of starting material.

Fig 5. Comparison of pepsin degradation of five substrate proteins (HRP, Rubisco LS, Hb, STI, and LTP).

On Panel A and Panel B, the amount of Coomassie Blue stained intact protein at each condition was quantified and is shown as a percentage relative to the amount of starting material. Panel A is at 10 U pepsin to 1 μg substrate protein and pH 1.2. Panel B is at 10 U pepsin to 1 μg substrate protein and pH 4. Panels C and D are SDS-PAGE analysis of pepsin degradation of LTP and STI, respectively, at pH 1.2 and 10 U pepsin to 1 μg substrate protein over time (0.5 to 60 minute(s)). The gel lanes are: 1: MW, 2: Pepsin Only, 3: Substrate Protein Only, 4: 0 minute: Pepsin + Substrate Protein, 5–11: 0.5, 2, 5, 10, 20, 30, 60 minute(s), respectively, of Pepsin + Substrate Protein.