Kinetic Properties of Human Erythrocyte Prolidase and Optimal Conditions for Prolidase Assay by Proline Colorimetric Determination

Ayşe Binnur Erbağcı; Yüksel Özdemir; Necat Yılmaz

doi:10.58600/eurjther.1999-10-1-2-1519-arch

Authors

Ayşe Binnur Erbağcı Department of Biochemistry and Clinical Biochemistry, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey
Yüksel Özdemir Department of Biochemistry and Clinical Biochemistry, Faculty of Medicine, University of Harran, Şanlıurfa, Turkey
Necat Yılmaz Department of Biochemistry and Clinical Biochemistry, Faculty of Medicine, University of Gaziantep, Gaziantep, Turkey

DOI:

https://doi.org/10.58600/eurjther.1999-10-1-2-1519-arch

Keywords:

Human erythrocyte, prolidase, kinetic properties, inhibition

Abstract

Prolidase (EC 3.4.13.9) is an iminodipeptidase that catalyses the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. it is involved in intracellular protein degradation, and apparently contributes to the conservation of iminoacides from endogenous and exogenous protein structures. Analysis of prolidase activity in human biological fluids and tissues has gained attention as a biochemical tool in acute and chronic liver diseases, adenocarcinoma of lungs and as a marker for fetal lung maturation and growth, in addition to long known genetic prolidase deficiency. in the present study, stability, activation and inhibition kinetics of human erythrocyte prolidase and optimal conditions for human erythrocyte prolidase assay were investigated. Level of proline, product of the enzymatic reaction was measured by Chinard's method. Human erythrocyte prolidase activity was stable tor six months at -10 °C. Preincubation of the enzyme at 37 °C with 1 mM MnCl2 provided appropriate activity.

Preincubation at 45 °C and 55 °C caused enzyme inactivation. After preincubation, prolidase activity was linearly related to incubation time up to at least two hours. Buffer ionic strength between 30-70 mM at pH 8.0 did not cause an obvious difference of prolidase activity. However pH changes between 7.0 and 8.5 caused significant differences with maximal activity at pH 8.0 The Km value tor Gly-L-pro dipeptide was approximately 7.0 mM. EDTA, iodo acetamide and some divalent metal ions (Cu2+, Fe2+, Zn2+, Hg2+) had inhibitory effect on human erythrocyte prolidase activity. According to our results, preincubation at 1 mM MnCl2 concentration for 2 hours at 37°C and incubation at 30 mM Gly-L-pro dipeptide concentration, 40 mM buffer ionic strength, pH 8.0 tor 30 minutes reveals optimal assay conditions for human erythrocyte prolidase. Presence of metal chelating agents and metal ions other than manganese in the reaction media can interfere results, thus should be avoided.

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