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:	Hüseyin Kayadibi, Burhanettin Bolat, Osman Metin İpçioğlu, Mustafa Gültepe, Ömer Özcan
Formato:	article
Lenguaje:	EN
Publicado:	De Gruyter 2007
Materias:	Prolidase photometric method proline inhibition Biochemistry QD415-436
Acceso en línea:	https://doaj.org/article/a88b1ebce0374ae08af2ad411b102cd8
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spelling	oai:doaj.org-article:a88b1ebce0374ae08af2ad411b102cd82021-12-02T13:51:34ZOptimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase0250-46851303-829Xhttps://doaj.org/article/a88b1ebce0374ae08af2ad411b102cd82007-05-01T00:00:00Zhttp://www.turkjbiochem.com/2007/012.pdfhttps://doaj.org/toc/0250-4685https://doaj.org/toc/1303-829XProlidase 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.Hüseyin KayadibiBurhanettin Bolat,Osman Metin İpçioğlu,Mustafa Gültepe,Ömer Özcan,De GruyterarticleProlidasephotometric methodproline inhibitionBiochemistryQD415-436ENTürk Biyokimya Dergisi, Vol 32, Iss 1, Pp 12-16 (2007)
institution	DOAJ
collection	DOAJ
language	EN
topic	Prolidase photometric method proline inhibition Biochemistry QD415-436
spellingShingle	Prolidase photometric method proline inhibition Biochemistry QD415-436 Hüseyin Kayadibi Burhanettin Bolat, Osman Metin İpçioğlu, Mustafa Gültepe, Ömer Özcan, Optimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase
description	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.
format	article
author	Hüseyin Kayadibi Burhanettin Bolat, Osman Metin İpçioğlu, Mustafa Gültepe, Ömer Özcan,
author_facet	Hüseyin Kayadibi Burhanettin Bolat, Osman Metin İpçioğlu, Mustafa Gültepe, Ömer Özcan,
author_sort	Hüseyin Kayadibi
title	Optimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase
title_short	Optimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase
title_full	Optimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase
title_fullStr	Optimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase
title_full_unstemmed	Optimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase
title_sort	optimization of the photometric enzyme activity assay for evaluating real activity of prolidase
publisher	De Gruyter
publishDate	2007
url	https://doaj.org/article/a88b1ebce0374ae08af2ad411b102cd8
work_keys_str_mv	AT huseyinkayadibi optimizationofthephotometricenzymeactivityassayforevaluatingrealactivityofprolidase AT burhanettinbolat optimizationofthephotometricenzymeactivityassayforevaluatingrealactivityofprolidase AT osmanmetinipcioglu optimizationofthephotometricenzymeactivityassayforevaluatingrealactivityofprolidase AT mustafagultepe optimizationofthephotometricenzymeactivityassayforevaluatingrealactivityofprolidase AT omerozcan optimizationofthephotometricenzymeactivityassayforevaluatingrealactivityofprolidase
_version_	1718392398668627968

Optimization of the Photometric Enzyme Activity Assay For Evaluating Real Activity of Prolidase

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