Colorimetric Assay

Purpose:

The colorimetric assay was used to determine whether our cloned enzymes were functional by testing their ability to cleave their respective substrates. These substrates were obtained from Sigma Aldrich (A1, A2, A3), Fisher Scientific (B1), and RPI (B2). They contain a 4-nitrophenol (pNP) group that turns yellow upon successful cleavage. The concentration of 4-nitrophenol can be quantified at 405 nm using a 96-well plate assay.

Experimental Design:

1. Qualitative assay

   Small-scale colorimetric tests were performed in PCR tubes with a total volume of 10 μL. Each tube contained 5 μL of 10 mM substrate stock solution, 1 μL of 0.1 μM enzyme, 1 μL of 10X GlycoBuffer 1 (500 mM sodium acetate, 50 mM CaCl2, pH 5.5), 1 μL of purified BSA (100 μg/mL). The remaining volume was adjusted with water to reach 10 μL. Reactions were incubated at 37 °C for 1 hour, then enzymatic activity was assessed by the development of yellow solution from pNP release.

2. Quantitative assay: Michaelis – Menten Kinetics Model

   A pNP standard curve was generated using concentrations from 80 µM to 10 mM, and a linear regression was performed to establish the relationship between pNP concentration and absorbance. This calibration allowed absorbance values from enzyme reactions to be converted into pNP concentrations, which were then used to calculate product formation over time.

   To quantify the enzyme activity, reactions were performed for our enzymes (A1, A2, A3, Cold-Adapted enzyme CA, B2) across a range of substrate concentrations (0.15625 mM, 0.3125 mM, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM). Each well contains 50 μL of substrate stock solution, 10 μL of 0.1 μM enzyme, 10 μL of 10X GlycoBuffer 1, and 30 μL of water. Absorbance at 405 nm was recorded every minute over a 3-hour period to track product formation. Each reaction was run in triplicate, and averaged results for each enzyme were used to calculate Michaelis – Menten constants for enzyme efficiency. The overall experiment was repeated in duplicate at 24°C.

   To analyze enzyme kinetics, the change in product concentration between t = 0 min and t = 10 min was calculated for each substrate concentration to determine the initial reaction rate (Vo). The 10-minute time point was selected to capture the initial linear phase of the reaction, where product formation reflects true enzyme activity before substrate depletion or saturation effects occur. These initial rates were then plotted against substrate concentration, and the data were fitted using least squares regression to generate Michaelis–Menten curves. From the fitted curve, Vmax (maximum reaction velocity) and Km (substrate affinity constant) were determined for each enzyme using Equation 1.

Porcine Blood Conversion and Crossmatch Hemagglutination Assay

Purpose:

The crossmatch porcine hemagglutination assay was designed to allow us to qualitatively assess the effectiveness of our enzymes to cleave antigens on blood. Human blood would be a more accurate model, but porcine blood was chosen as an alternative to remain within safety limitations. Porcine blood does not contain the type B antigen, but the enzyme, a-galactosidase, that targets the B antigen chain also cleaves alpha-gal, which is present in porcine blood. Humans universally have an immune reaction to alpha-gal, so this allows us to assess how active our enzymes are.

While there is not extensive literature on the existence of antigens that extend from the A and a-gal antigens on porcine blood, pigs also express the genes that are responsible for the extended antigens in humans:

• FUT1/FUT2 is responsible for H Type 3 (Targeted by A3)
• B3GALNT1 is responsible for ExtB (Targeted by B2)

Thus, we treated A Type red blood cells (RBCs) with A1, A1-3, and all enzymes, respectively to assess change in agglutination. O Type Blood was also treated with B1-2, and crossmatched with human serum.

Experimental Design:

The procedure for blood conversion was adapted from the 2023 paper by Jensen et al [3]. Type A and Type O porcine blood from Lampire Biological Laboratories was centrifuged to separate blood components, processed into packed red blood cells, and diluted in a saline conversion buffer (200 mM glycine, 3 mM NaCl adjusted to pH 6.8). From the centrifuged blood, type O porcine plasma was also collected and used to test for the presence of A antigens on A type Blood, since it contains anti-A antibodies. To test for the presence of alpa-gal, commercial human AB serum was used, as all human serum contains antibodies against alpha-gal (Sigma Aldrich).

1. Tube Test Crossmatch Hemagglutination

   A tube test which involves grading reactions based on observable agglutinates was utilized to assess cleavage of antigens on blood before and after conversion. Normal and microscopic pictures were taken of these reactions.

2. EldonCard

   The EldonCard, a commercially available dry blood-typing card, was used to detect A and B antigens. All cards contain anti-A, anti-B, and control fields. An anti-D field is also included to detect positive and negative types, which is not on porcine blood.

Enzyme treatments and their expected outcomes are shown in the table below:

Protocols

Common Wet Lab Protocols

Protein Purification Protocols

Colorimetric Assay Protocols

Porcine Blood Conversion and Crossmatch Assay Protocols

References

[1] Michaelis-Menten Kinetics. (2013, October 2). Chemistry LibreTexts. https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_ Modules_(Biological_Chemistry)/Enzymes/Enzymatic_Kinetics/Michaelis-Menten_Kinetics

[2] Ricci Hagman, J. (2023). Atypical Carbohydrate Blood Groups and Their Removal: Implications in Transfusion Medicine. [Doctoral Thesis (compilation), Department of Laboratory Medicine]. Lund University, Faculty of Medicine.

[3] Jensen, M., Stenfelt, L., Ricci Hagman, J. et al. Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood. Nat Microbiol 9, 1176–1188 (2024). https://doi.org/10.1038/s41564-024-01663-4