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

<div><p>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 <i>in vitro</i> 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 <i>in vitro</i> 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 <i>in vitro</i> 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 <i>in vitro</i> 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.</p></div

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

<p>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.</p

Five test proteins represent diverse characteristics of physiochemical properties that could affect pepsin hydrolysis.

<p>Five test proteins represent diverse characteristics of physiochemical properties that could affect pepsin hydrolysis.</p

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

<p>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.</p

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

<p>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.</p