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Isolation of Prolidase from Amniotic Fluid and Study of its Kinetic and Affinity Properties Towards Pharmacological Compounds | ||
| Journal of Education and Science | ||
| Article 6, Volume 28, Issue 3, 2019, Pages 52-72 PDF (1.33 M) | ||
| DOI: 10.33899/edusj.2019.162958 | ||
| Author | ||
| Luay Aabd Al-Helaly | ||
| Abstract | ||
| The research included the isolation of prolidase from human amniotic fluid using different biochemical techniques, One proteinous peak had been isolated by gel filtration using sephadex )G-50) and from sephadex (G-100) that produced by ammonium sulphate precipitation (60%) after dialysis. The approximately molecular weight of the enzyme using gel filtration chromatography (G-100) was (52269.7) Dalton and specific activity of 13516.6 unit/mg protein with 75 fold of purification. The results showed that the optimum conditions of purified enzyme from amniotic fluid were at (50 µg/ml) of protein as a source of the enzyme using (60 mM/L) Tris-HCl buffer solution at pH (8.0) act for (32) minutes at (39°C). Using Line Weaver-Burk plot, the values of maximum velocity (Vmax) and Michaelis constant (Km) were found to be (3448.2 U/L) and (10 mM/L) respectively using Gly-Pro as a substrate. Finally, also, involved the study of the effect pharmacological chemicals compounds on the enzyme activity, the results showed that the drug chemical compounds for type ceramide, metoclopramide, pseudoephedrine, diphenhydramine-HCl, chloramphnicol, paracetamol and allopurinol give the inhibition type competitive, with increased in Km value to 66.6, 71.42, 52.63, 55.55, 83.33, 90.9 and100.0 mM/L respectively for inhibitors above, while the others pharmacological compounds for theophlline anhydrous, caffeine anhydrous, metronidazole and chlorpheniramine maleate, give the inhibition type of non-competitive with decreased in Vmax values to 2173.9, 2439.0, 2000.0 and 2325.58 U/L respectively, but dexathamazone was an activator to the prolidase approximately 27.18 U/L. | ||
| Keywords | ||
| Isolation; enzyme; Prolidase; Amniotic fluid; inhibitors; Activators | ||
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