Purification and characterization of malate dehydrogenase from sheep liver (Ovis aries): Application in AST assay diagnostic kit

Doaa B Darwish ,Mohamed M Abdel-Monsef ,Mohamed Helmy ,Hassan Masoud ,Mahmoud A A Ibrahim

doi:10.7324/japs.2018.8216

Abstract

Malate dehydrogenase enzyme is a major component in aspartate aminotransferase (AST) diagnostic kit that used in diagnosis and monitoring of liver diseases. This study aimed at purification and characterization of MDH enzyme from sheep liver for direct application in preparation of AST kit. Two malate dehydrogenase isoenzymes were resolved from sheep liver by chromatography on DEAE-cellulose column, one major sheep liver MDH (SLMDH) and another minor one. SLMDH was extracted and purified by ammonium sulfate fractionation and chromatographical separation on anion exchanger and gel filtration resins. The purified sheep liver MDH specific activity is 6.7 units / mg protein representing 11.6 purification folds and 27.7 % yield. The molecular mass of SLMDH intact protein is 68 ± 1.6 kDa. SLMDH turned out to be homogeneous and consists of two identical subunits of 34 ± 1.2 kDa each. The SLMDH isoelectric point (pI) value is estimated at pH 6.2 and displayed its optimum activity at pH 9.6 and has Km value of 1.4 mM for NAD and 11 mM for malate. FeCl2, NiCl2 and ZnCl2 inhibited the SLMDH activity. The purified SLMDH is applied in the preparation of AST diagnostic kit that found sensitive and comparable with a commercially available one.

Highlights

Malate dehydrogenase (MDH: EC 1.1.1.37) is widely distributed enzyme through living organisms that present in animals, plants and microorganisms and it is an essential enzyme in the central oxidative pathway
The purification procedure of MDH from the sheep liver is very convenient since it involved ammonium sulfate fractionation, anion-exchanger DEAEcellulose resin chromatography and size-exclusion chromatography on Sephacryl S-300 resin
Different MDH purification methods were stated from rat liver by ion-exchange chromatography with affinity elution by NADH (Wiseman et al, 1991) and from chicken liver by ammonium sulfate precipitation, affinity chromatography on 5’AMP-Sepharose and Blue-Sepharose CL-6B columns (Gelpi et al, 1988)

Summary

Introduction

Malate dehydrogenase (MDH: EC 1.1.1.37) is widely distributed enzyme through living organisms that present in animals, plants and microorganisms and it is an essential enzyme in the central oxidative pathway. MDH is a catalyst in the oxidation process of malate to oxaloacetate and uses the reduced NAD+ to NADH as a cofactor. MDH is one of the main enzymes in the tricarboxylic acid cycle (TCA) that performs significant metabolic part in aerobic energy producing pathways and in malate shuttle. Malate dehydrogenases were found in plants and some eukaryotes in different organelles as glyoxysomes, chloroplasts and peroxisomes (Gietl, 1992). In aerobic prokaryotic and eukaryotic organisms, the TCA cycle is the main oxidative

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