Optimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase
Prolidase cleaves the bonds of dipeptides containing proline (X-Pro) and an importantenzyme in collagen metabolism. Prolidase activity is generally determinedby photometric methods based on the measurement of proline levels produced byprolidase. We aimed to investigate the measured prolidase activit...
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| Autores principales: | , , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
De Gruyter
2007
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/a88b1ebce0374ae08af2ad411b102cd8 |
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| Sumario: | Prolidase cleaves the bonds of dipeptides containing proline (X-Pro) and an importantenzyme in collagen metabolism. Prolidase activity is generally determinedby photometric methods based on the measurement of proline levels produced byprolidase. We aimed to investigate the measured prolidase activity is suitable forthe assessment of its physiological activity. Effects of manganese on the enzymeactivation, protein precipitation and reading steps of the photometric method, andinhibitory effect of proline on the enzyme were analyzed. The intra- and inter-assayCVs % were higher than 10 % for photometric method and turnaround timewas 6-8 hours/test. The activation reagent containing manganese was not stableand its concentration was not optimal for enzyme activation. Thus we modifiedthe photometric method by changing manganese concentrations and pH of activatingsolution, eliminating protein-precipitating step, arranging the pH of colorreagent to the pH optimum for ninhidrin reaction and shortened incubation timewith substrate. The inhibitory effect of proline on the prolidase activity even in thephysiological and produced proline concentrations during enzymatic analysis maylimit the analytical performance of prolidase assays. In conclusion, the modifiedphotometric method presented in this study seems to be more reliable than the classicalphotometric method and measured prolidase activity may not reflect the truephysiological activity of enzyme due to proline inhibition. |
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