Purification and Characterisation of Lactate Dehydrogenase: An Undergraduate Biochemistry Laboratory Experiment
Advances in Biochemistry
Volume 2, Issue 1, February 2014, Pages: 14-23
Received: Feb. 2, 2014;
Published: Feb. 20, 2014
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Yannis Karamanos, Laboratoire de Biochimie, Faculté des Sciences Université d’Artois, Lens, France
The practical work described here was designed in the aim of combining several periods that were previously carried-out independently during the academic year and to more appropriately mimic a "research" environment. It illustrates several fundamental biochemical principles as well as experimental aspects and important techniques including spectrophotometry, chromatography, centrifugation, and electrophoresis. Lactate dehydrogenase (LDH) is an enzyme of choice for a student laboratory experiment. This enzyme has many advantages, namely its relative high abundance, high specific activity and high stability. In the first part, the purification scheme starting from pig heart includes ammonium sulphate fractionation, desalting by size exclusion chromatography, anion exchange chromatography and pseudo-affinity chromatography. In the second part of the work the obtained fractions are accessed for protein and activity content in order to evaluate the efficiency of the different purification steps, and are also characterised by electrophoresis using non-denaturing and denaturing conditions. Finally, in the third part, the purified enzyme is subjected to comprehensive analysis of its kinetic properties and compared to those of a commercial skeletal muscle LDH preparation. The results presented thereafter are representative of the data-sets obtained by the student-pairs and are comparable to those obtained by the instructors and the reference publications. This multistep purification of an enzyme from its source material, where students perform different purification techniques over successive laboratory days, the characterisation of the purified enzyme, and the extensive approach of enzyme kinetics, naturally fits into a project-based biochemistry learning process.
Purification and Characterisation of Lactate Dehydrogenase: An Undergraduate Biochemistry Laboratory Experiment, Advances in Biochemistry.
Vol. 2, No. 1,
2014, pp. 14-23.
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