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
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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.
Lactate Dehydrogenase, LDH, Protein Purification, Enzymes, Laboratory Experiment
To cite this article
Yannis Karamanos, Purification and Characterisation of Lactate Dehydrogenase: An Undergraduate Biochemistry Laboratory Experiment, Advances in Biochemistry. Vol. 2, No. 1, 2014, pp. 14-23. doi: 10.11648/j.ab.20140201.13
A.J. Turner, A simple a colourful procedure to demonstrate the principles of affinity chromatography, Biochem. Educ., 7 (1979) 60–61.
E.C. Wolf, The Partial Purification and Characterization of Lactate Dehydrogenase, Biochem. Educ., 16 (1988) 231–234.
R.J. Gay, R.B. McComb, G.N. Bowers, Optimum reaction conditions for human lactate dehydrogenase isoenzymes as they affect total lactate dehydrogenase activity, Clin. Chem., 14 (1968) 740–753.
K.L. Manchester, Kinetics of lactate deshydrogenase: a textbook problem, Biochem. Educ., 5 (1977) 15.
O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randanll, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265–275.
A.B. Coleman, New ideas for an old enzyme: A short, question-based laboratory project for the purification and identification of an unknown LDH isozyme, Biochem. Mol. Biol. Educ., 38 (2010) 253–260.
U.K. Laemmli, Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4, Nature, 227 (1970) 680–685.
P.H. Springell, T.A. Lynch, Staining artifacts following electrophoretic separation of LDH isozymes, Anal. Biochem., 74 (1976) 251–253.
R. Stambaugh, D. Post, Substrate and product inhibition of rabbit muscle lactic dehydrogenase heart (H4) and muscle (M4) isozymes, J. Biol. Chem., 241 (1966) 1462–1467.
J.L. Powers, N.E. Kiesman, C.M. Tran, J.H. Brown, V.L.H. Bevilacqua, Lactate Dehydrogenase Kinetics and Inhibition Using a Microplate Reader, Biochem. Mol. Biol. Educ., 35 (2007) 287–292.
K.L. Manchester, LDH: Plus ça change, plus c’est la même chose Continuing problems with textbook presentations of the kinetic properties of the isozymes of lactate dehydrogenase, Biochem. Educ., 22 (1994) 91–93.
T. Wuntch, R.F. Chen, E.S. Vesell, Lactate dehydrogenase isozymes: kinetic properties at high enzyme concentrations, Science, 167 (1970) 63–65.
T. Wuntch, E.S. Vesell, R.F. Chen, Studies on rates of abortive ternary complex formation of lactate dehydrogenase isozymes, J. Biol. Chem., 244 (1969) 6100–6104.
A. Pesce, T.P. Fondy, F. Stolzenbach, F. Castillo, N.O. Kaplan, The comparative enzymology of lactic dehydrogenases 3 Properties of the H4 and M4 enzymes from a number of vertebrates, J. Biol. Chem., 242 (1967) 2151–2167.
S. de S. Groth, R. Webster, A. Datyner, Two new staining procedures for quantitative estimation of proteins on electrophoretic strips, Biochim. Biophys. Acta, 71 (1963) 377–391.
R. Boyer, Concepts and skills in the biochemistry/molecular biology lab, Biochem. Mol. Biol. Educ., 31 (2003) 102–105.
B. Alberts, Redefining science education, Science, 323 (2009) 437.
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