GB 502 Experimental Biotechnology For Malate Dehydrogenase

Answer:

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 resin.

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 activity.

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

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 Law 

With the use of the Molar Extinction Coefficient of NADH

=at 340 nm

-0.0046= 

This means the quantity of NADH that was adopted in the enzyme assay was

The activity of the enzyme per mL was determined. The enzyme activity was established to be  when the enzyme extract amount used was 0.1 mL.

This enables equating both sides

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 preparation.

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)