New ideas for an old enzyme: A short, question‐based laboratory project for the purification and identification of an unknown LDH isozyme

Abstract

Enzyme purification projects are an excellent way to introduce many aspects of protein biochemistry, but can be difficult to carry out under the constraints of a typical undergraduate laboratory course. We have designed a short laboratory project for the purification and identification of an ''unknown'' lactate dehydrogenase (LDH) isozyme that can fit into a multiproject course without consuming too many laboratory days. The streamlined purification utilizes ammonium sulfate precipitation, affinity chromatography, and size exclusion chromatography to give good recovery of LDH with minimal equipment requirements, and can be completed in three laboratory periods of 3-4 hours. As part of this, we have designed a novel, qualitative format for an LDH activity assay that allows students to rapidly screen their column chromatography fractions without the need of a spectrophotometer or plate reader. The analysis phase of the project is question-driven, and can be completed in two laboratory periods. The students must determine which purification technique was most effective by quantifying LDH activity and total protein content at each step of the purification, and then identify their unknown isozyme through agarose gel electrophoresis. This module provides an engaging format for teaching protein biochemistry, with the flexibility to allow an instructor to modify it for their particular curriculum.

Purification and Characterisation of Lactate Dehydrogenase: An Undergraduate Biochemistry Laboratory Experiment

“…As it was recently pointed out [6], hands-on experience with performing experiments and analyzing data helps students to develop problem-solving skills that will benefit them throughout their life. And according to a popular editorial in Science [17] such problem-solving skills are not being adequately taught to undergraduate students, the result of too much focus on presenting scientific explanations without covering the scientific processes and reasoning.…”

“…LDH screening assay (spot test). This quick evaluation of the presence of LDH activity using a 96-well plate was adapted from [6]. Creation of NADH in the presence of LDH drives the successive reduction of phenazine methosulphate (PMS) and then p-iodo nitrotetrazolium (INT).…”

Profile of Students' Creative Thinking Skills on Quantitative Project-Based Protein Testing using Local Materials

“…Some other studies have also found the relationship of creative thinking skills to experimental activities in the lab. Experimental activities can make students do a practicum with hands on as well as minds on activity (Bellin, Bruno, and Farrow, 2010;Coleman, 2010;Witherow and Carson, 2011;Trapani and Clarke, 2012).…”

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

“…We chose the enzyme lactate dehydrogenase (LDH) as the target molecule for isolation and electrophoretic characterization. While previous papers − and laboratory manuals , have described the isolation of this protein from mammalian heart or skeletal muscle, we chose to do the isolation from mammalian blood, a nontraditional source that offers a number of advantages. (1) Red blood cells (RBC) are easily isolated in pure form from whole blood by repeated centrifugation in isotonic saline; (2) the cytosolic fraction, or membrane-free lysate, is generated by simple hypotonic lysis followed by centrifugation; (3) the resulting lysate yields LDH activity that is stable for several weeks, allowing a measure of flexibility with regard to the timing of its preparation for the class; (4) the lack of any significant protease activity in the lysate alleviates the need to add expensive and toxic protease inhibitors; (5) the fold purification and percentage recovery of activity are far superior to those typically reported for the isolation of LDH using mammalian muscle tissues. − …”