Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies
Abstract
:1. Introduction
2. Results and Discussion
2.1. Preparation and Characterization of POPC Liposomes and Mixed Micelles
2.2. Preparation of Standard Absorption Curve of Bromothymol Blue (BTB)
2.3. Effect of CaCl2 on sPLA2 Activity
2.4. Effect of pH on PLA2 Activity
2.5. Comparison of PLA2 IIA Kinetics on POPC-Based Self-Assemblies (Liposomes vs. Mixed Micelles)
2.6. Comparison of PLA2 IIA and IB Assay Kinetics with POPC-Based Liposomal Vesicles and Mixed Micelles
2.7. Inhibition Study of PLA2 IIA Using Varespladib
3. Materials and Methods
3.1. Materials
3.2. Methods
3.2.1. Preparation of Buffers and Stock Solutions
3.2.2. Preparation of 1.0 mM POPC Liposomes via Extrusion Method
3.2.3. Preparation of POPC Mixed Micelles with Triton X-100 via Sonication Method
3.2.4. Preparation of Standard Curve Using Bromothymol Blue (BTB)
3.2.5. Effect of Ca2+ Ion on PLA2 Activity
3.2.6. Effect of pH on PLA2 Activity
3.2.7. Assay Method for PLA2 Kinetic Study Using POPC-Based Liposomes and Mixed Micelles
3.2.8. PLA2 Inhibition Study with the Developed Assay Using a Standard Inhibitor Varespladib
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Formulations | Z.avg (nm) | Main Peak (nm) | PDI | %Volume |
---|---|---|---|---|
POPC pure (liposomal) | 113.3 | 108.4 | 0.10 | 100% |
Tx:POPC 2:1 mixed micelles | 70.0 | 50.3 | 0.22 | >95% |
Tx:POPC 1:1 mixed micelles | 63.1 | 32.0 | 0.37 | >98% |
Tx:POPC 1:2 mixed micelles | 63.7 | 33.4 | 0.41 | >98% |
Tx:POPC 1:6.67 mixed micelles | 108.8 | 30.4 | 0.50 | >90% |
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Khan, S.A.; Ilies, M.A. Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies. Int. J. Mol. Sci. 2024, 25, 9517. https://doi.org/10.3390/ijms25179517
Khan SA, Ilies MA. Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies. International Journal of Molecular Sciences. 2024; 25(17):9517. https://doi.org/10.3390/ijms25179517
Chicago/Turabian StyleKhan, Shibbir Ahmed, and Marc A. Ilies. 2024. "Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies" International Journal of Molecular Sciences 25, no. 17: 9517. https://doi.org/10.3390/ijms25179517