Purification and Electrophoretic Characterization of Lactate Dehydrogenase from Mammalian Blood: A Different Twist on a Classic Experiment

Abstract

A multiweek protein purification suite, suitable for upper-division biochemistry or biotechnology undergraduate students, is described. Students work in small teams to isolate the enzyme lactate dehydrogenase (LDH) from a nontraditional tissue source, mammalian blood, using a sequence of three column chromatographic procedures: ion-exchange, size exclusion, and affinity chromatography. Protein and enzyme activity elution profiles are determined by graphical analysis of assay data collected using rapid microplate spectrophotometric assays. Students perform quantitative assays on LDH pools and use these data to build a purification table for use in evaluating the protocol. The protocol typically generates final overall fold-purifications from 1500 to 2500 and activity recoveries of 45–60%. Electrophoretic separations in both denaturing and native-gel format are analyzed both visually and by use of commercial digital analysis software to assess the isoenzyme pattern of isolated LDH and to further evaluate the purification. Assessment of student work revealed a high level of achievement of course learning goals that include development of critical thinking skills required to (1) critically evaluate an experimental protocol and (2) draw conclusions based on a variety of forms of experimental data.

Learning about Fluorescence in Undergraduate Biochemistry: Enzyme Kinetics Using a Low-Cost, Student-Built Fluorescence Spectrometer

“…However, NADH is also well-known to fluoresce at 460 nm, and its fluorescent properties have also been previously exploited to monitor reaction progress. − Lactate dehydrogenase is a nearly ubiquitous enzyme that catalyzes the interconversion of L-lactate and pyruvate, with simultaneous interconversion of NAD + and NADH (Scheme ). Previous reports use lactate dehydrogenase in the biochemistry lab to teach purification and specific activity, , and this new exercise could immediately follow that work. Six weeks of our first semester biochemistry laboratory sequence involves the extraction, purification, and characterization of lactate dehydrogenase from domesticated chicken breast ( Gallus gallus ).…”