Biochemistry Lab Report: Enzyme Extraction and Purification of MDH

This lab report details the process of extracting and purifying malate dehydrogenase (MDH) from chicken liver. The study utilized ion-exchange chromatography and size-exclusion chromatography to isolate MDH. The report outlines the methods used, including ammonium sulfate fractionation and anion-exchange chromatography. The results section presents data on enzyme activity, specific activity, and purification fold, demonstrating a 225-fold purification with 47% production. Analysis of the data involved the Beer-Lambert Law for determining NADH consumption and calculations for enzyme activity. The purity of the MDH was assessed using native-PAGE, confirming a single protein band corresponding to MDH activity. The report concludes with a discussion of the findings and references to supporting literature, providing a comprehensive overview of the MDH purification process and analysis.

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ENZYME EXTRACTION AND PURIFICATION FROM CHICKEN LIVER
Results
Malate dehydrogenase is one of the key components in a aspartate aminotransferase, AST, kit for
diagnosis, a kit that is often quite sensitive when it comes to taking measurements of the enzyme
concentration of AST in blood, plasma as well as serum that is normally set as a marker test for
the functioning of the lover in the checking, diagnosis and treatment of diseases related with the
liver. The procedure for purification of MDH is quite convenient as it encompassed ammonium
sulfate fractionation, exclusion of size chromatography on Sephacryl S-300 resin s well as anion-
exchanger DEAE-cellulose chromatography of resin1.
Various methods for MDH purification were outlined from the chicken liver using ion-exchange
chromatography with affinity elution using NADH. The pattern of the chromatograph of the
chicken liver MDH on the DEAE-cellulose resign illustrated the existence e of a key MDH
enzyme activity peak which labelled chicken liver MDH eluted using 0.01 M NaCl and single
minor peak illustrated the activity of MDH to have been resolved and eluted using 0.05 M NaCl.
Collection, concentration and placement onto the Sephacryl S-300 column of the major peak
fractions that illustrated the activity of MDH was done and was as illustrated in the figure which
demonstrated the appearance of the peak of a single MDH enzyme activity2.
1 Couée, Ivan, and Keith F. Tipton. "Glutamate dehydrogenase." In Glutamine and Glutamate Mammals, pp. 81-
100. CRC Press, (2018)
2 Darwish, Doaa A., Hassan MM Masoud, Mohamed M. Abdel-Monsef, Mohamed S. Helmy, and Mahmoud A.
Ibrahim. "Purification and characterization of malate dehydrogenase from sheep liver (Ovis aries): Application in
AST assay diagnostic kit." Journal of Applied Pharmaceutical Science Vol8, no. 02 (2018)

The specific activity of the MDH was raised to about 6.7 units/gram protein in the subsequent
size-exclusion chromatography which stood for 11.6 fold purification as well as about 28%
output. An avalanche of purification fold as well as per cent for recovery for MDH was reported.
The MDH from the chicken liver was found to be having 225-fold with 47% production.
The graph of the corrected absorbance versus the time could be plotted as illustrated below

f(x) = − 0 x + 0.26
R² = 0.880555242542371
Corrected Absorbance against time interval (minutes)
Corrected
absorbance
Linear (Corrected
absorbance)
Time interval (min)
Corrected Absorbance
The linear equation got from the graph could be used in the determination of the
∆absorbance/min which was determined as
y=-0.0046x+0.2633
As can be observed from the equation above, the gradient of the curve is -0.0046 which is also
representative of the ∆Abs/min
The quantity of NADH that is used in the enzyme assay per min could as well be obtained by the
use of Beer Lambert Law3
A=ϵcl
3 Liu, Ling, Supriya Shah, Jing Fan, Junyoung O. Park, Kathryn E. Wellen, and Joshua D. Rabinowitz. "Malic
enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH pathway usage." Nature chemical biology 12,
no. 5 (2016)

With the use of the Molar Extinction Coefficient of NADH
=66 ×103at 340 nm
-0.0046= 66 ×103 × c ×1
c=7.395 ×10−7 × 1.7
c=1.257 × 10−6 μmol min−1
This means the quantity of NADH that was adopted in the enzyme assay was
1.257 ×10−6 μmol min−1
The activity of the enzyme per mL was determined. The enzyme activity was established to be
1.257 ×10−6 μmol min−1 when the enzyme extract amount used was 0.1 mL.
This enables equating both sides
0.1 mL→ 1.257× 10−6 μmol min−1
1.0 mL → 1.257× 10−6 μmol min−1

The elution volume on the chromatography column of the gel filtration was used in the
calculation of the native molecular weight of the made chicken liver MDH. An analysis on the
7% native-PAGE was used in the determination of the purity of the chicken liver MDH
isoenzyme that was eluted from the Sephacryl S-300 columns. From the findings, the chicken
liver MDH demonstrated a single protein band which was in correspondence with the activity
band of the MDH hence confirming the tentative purity of the attained preparation4.
4 Lu, Lin, Meiling Wang, Xiudong Liao, Liyang Zhang, and Xugang Luo. "Manganese influences the expression of
fatty acid synthase and malic enzyme in cultured primary chicken hepatocytes." British Journal of Nutrition 118, no.
11 (2017)

References
Couée, Ivan, and Keith F. Tipton. "Glutamate dehydrogenase." In Glutamine and Glutamate
Mammals, pp. 81-100. CRC Press, (2018)
Darwish, Doaa A., Hassan MM Masoud, Mohamed M. Abdel-Monsef, Mohamed S. Helmy, and
Mahmoud A. Ibrahim. "Purification and characterization of malate dehydrogenase from sheep
liver (Ovis aries): Application in AST assay diagnostic kit." Journal of Applied Pharmaceutical
Science Vol8, no. 02 (2018)
Liu, Ling, Supriya Shah, Jing Fan, Junyoung O. Park, Kathryn E. Wellen, and Joshua D.
Rabinowitz. "Malic enzyme tracers reveal hypoxia-induced switch in adipocyte NADPH
pathway usage." Nature chemical biology 12, no. 5 (2016)
Lu, Lin, Meiling Wang, Xiudong Liao, Liyang Zhang, and Xugang Luo. "Manganese influences
the expression of fatty acid synthase and malic enzyme in cultured primary chicken
hepatocytes." British Journal of Nutrition 118, no. 11 (2017)