Purification of thioredoxin reductase from Spirulina platensis by affinity chromatography and investigation of kinetic properties

by Selleckchem | Feb 05 2024

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Spirulina platensis, which is one of the blue-green algae in the form of spiral rings, belongs to the cyanobacteria class. Spirulina platensis can produce Trx under stress conditions. If it can produce Trx, it also has TrxR activity. Therefore, in this study, the TrxR enzyme was purified for the first time from Spirulina platensis, an algae the most grown and also used as a nutritional supplement in the world. A two-step purification process was used: preparation of the homogenate and 2',5'-ADP sepharose 4B affinity chromatography. The enzyme was purified with a purification fold of 1059.51, a recovery yield of 9.7 %, and a specific activity of 5.77 U/mg protein. The purified TrxR was tested for purity by SDS-PAGE. The molecular weight of its subunit was found to be about 45 kDa. Optimum pH, temperature and ionic strength of the enzyme were pH 7.0, 40 °C and 750 mM in phosphate buffer respectively. The Michaelis constant (Km) and maximum velocity of enzyme (Vmax) values for NADPH and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) are 5 μM and 2.2 mM, and 0.0033 U/mL and 0.0044 U/mL, respectively. Storage stability of the purified enzyme was determined at several temperatures. The inhibition effects of Ag+, Cu2+, Al3+ and Se4+ metal ions on the purified TrxR activity were investigated in vitro. While Se4+ ion increased the enzyme activity, other tested metal ions showed different type of inhibitory effects on the Lineweaver-Burk graphs.

Comments:

It seems like you've provided a detailed overview of a study on the purification and characterization of thioredoxin reductase (TrxR) from Spirulina platensis. Here's a breakdown of the key points:

1. **Thioredoxin System:** Comprised of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Spirulina platensis, a type of blue-green algae, can produce Trx under stress conditions, suggesting the presence of TrxR activity.

2. **Study Objective:** The study aimed to purify TrxR from Spirulina platensis, a widely cultivated algae used as a nutritional supplement.

3. **Purification Process:** Used a two-step purification process involving homogenate preparation and 2',5'-ADP sepharose 4B affinity chromatography. Achieved a high purification fold of 1059.51, a recovery yield of 9.7%, and a specific activity of 5.77 U/mg protein.

4. **Characterization:**
   - SDS-PAGE analysis indicated a subunit molecular weight of about 45 kDa for the purified TrxR.
   - Optimal conditions for the enzyme were pH 7.0, 40 °C temperature, and 750 mM ionic strength in a phosphate buffer.
   - Determined Michaelis constant (Km) and maximum velocity (Vmax) values for NADPH and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB).
   - Evaluated storage stability at various temperatures.

5. **Inhibition and Activation Effects:** Investigated the impact of Ag+, Cu2+, Al3+, and Se4+ metal ions on TrxR activity.
- Se4+ increased enzyme activity, while other tested metal ions exhibited inhibitory effects, showing varied patterns on the Lineweaver-Burk graphs.

This study's findings contribute to understanding the purification, characterization, and behavior of TrxR from Spirulina platensis, shedding light on its potential applications and interactions with different metal ions.