1. Purpose and Scope
Purpose: To quantitatively evaluate, under controlled conditions, the activity retention, structural integrity, and aggregation risk of recombinant enterokinase (rEK) during exposure at 25 °C, and to define acceptable exposure duration and influencing factors.
Scope:
- Applicable to research-grade materials and in-process intermediates (stock solution, reconstituted from lyophilized form, buffers containing salt or glycerol).
- Finished enzyme preparations may refer to this procedure, but final judgment should be made in conjunction with their established quality or release specifications.
- Can be used to assess: room-temperature exposure tolerance, impact of deviations (short-term room temperature), transportation, and online standby conditions.
- For samples containing strong inhibitors, complexes of enzymes, or requiring buffers other than pH 8.0, separate verification is recommended.
2. Principle and Activity Evaluation
Inactivation and aggregation mechanisms: During exposure at 25 °C, rEK may undergo
(1) decreased catalytic efficiency due to conformational loosening;
(2) autolysis/limited proteolysis leading to damage of the active site;
(3) interface-induced or heat-induced aggregation;
These changes are usually reflected as reduced conversion, appearance of self-degradation bands of the enzyme on SDS-PAGE, or high–molecular weight aggregates remaining at the top of the gel.
Activity evaluation: rEK is a serine protease that recognizes the DDDDK↓X site. Add an equal amount of enzyme to a fixed amount of protein substrate; after reaction at 25 °C for 14–16 h, compare the band densities of substrate and cleavage products by SDS-PAGE, and calculate conversion (% conversion) as the relative activity readout; normalize to the ice-kept t = 0 control to obtain relative activity retention (%).
3. Reagents
Reagent A: 25 mM Tris-HCl buffer, pH 8.0 (25 °C)
Preparation: Weigh 0.303 g Tris (free base), dissolve in ~80 mL ultrapure water (25 °C), adjust to pH 8.0 with 2 M HCl, then bring to volume to 100 mL with water.
Reagent B: Recombinant enterokinase
Storage: −20 °C (avoid repeated freeze–thaw cycles; keep on ice during use).
Reagent C: Substrate solution (protein substrate)
Storage: −80 °C (aliquoting recommended to avoid freeze–thaw).
4. Procedures
4.1 Sample preparation
- Remove recombinant enterokinase (rEK) from −20 °C and place immediately on ice to thaw slowly.
- Check whether the solution is clear, without obvious precipitate or flocculent material; if slight precipitate is present, centrifuge at 4 °C for 5 min (10,000×g) and use the supernatant.
- Remove the substrate solution (Reagent C) from −80 °C, thaw on ice, and mix gently.
- Use prechilled pipette tips throughout; avoid generating bubbles.
4.2 Dilution and exposure
- Take an appropriate amount of rEK solution and dilute with Reagent A to a working concentration of 0.1 U/μL.
- Aliquot into multiple 1.5 mL microcentrifuge tubes (each tube containing enough for one enzyme activity test, ~20–50 μL), and label as Day 0, 1, 2, 3, 5, 7.
- Place all samples in a 25 °C incubator or thermostatic water bath for exposure; keep the Day 0 sample on ice throughout as a control.
- At each set time point (Days 1, 2, 3, 5, 7), immediately remove the corresponding tube and place on ice for later use.
4.3 Enzymatic digestion reaction
- Take 1 μL of rEK solution (0.1 U/μL) and add to 50 μL of substrate solution (each tube contains 50 μg substrate at 1 mg/mL).
- Mix gently without vortexing; briefly spin down to collect liquid at the bottom.
- Incubate at 25 °C for 14–16 h.
- At the end of the reaction, add an equal volume of 2× SDS-PAGE loading buffer and heat at 95 °C for 5 min to terminate the reaction.
4.4 SDS-PAGE analysis
- Load 10 μL per lane onto a 12% resolving gel.
- Electrophoresis conditions: constant voltage 120 V, ~1 h.
- Staining and destaining: stain with Coomassie Brilliant Blue R-250 for 1 h and destain until the background is clear.
- Photograph and use image analysis software (e.g., ImageJ) to measure the grayscale values of substrate and cleavage product bands.
- Calculate conversion (% conversion) = [cleavage product / (substrate + cleavage product)] × 100%.
- Set the conversion of the t = 0 on-ice sample as 100% and calculate the relative activity retention.
5. Notes and Optimization Recommendations
- For each measurement, set a t = 0 on-ice control and a historical positive control; compare samples from the same batch on the same gel as much as possible.
- Keep the substrate batch, loading amount, gel and development conditions consistent; record room temperature and exposure time.
- Handle gently and avoid foaming; use low-binding consumables to reduce interfacial contact.
- Strictly control pH 8.0 and ionic strength; keep the enzyme/substrate ratio and reaction time constant (0.1 U : 50 μg, 14–16 h).
- Aliquot and freeze for storage to avoid repeated freeze–thaw; keep samples on ice throughout experimental intervals.