New Ideas for an Old Enzyme: A Short, Question-based Laboratory Project for the Purification and Identification of an Unknown LDH Isozyme*

Transcription

1 Q 2010 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 38, No. 4, pp , 2010 Laboratory Exercises New Ideas for an Old Enzyme: A Short, Question-based Laboratory Project for the Purification and Identification of an Unknown LDH Isozyme* Received for publication, August 31, 2009, and in revised form, November 23, 2009 Aaron B. Coleman From the Division of Biological Sciences, University of California, San Diego, La Jolla, California 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. Keywords: Problem-based learning, enzymes and catalysis, laboratory exercises, lactate dehydrogenase, protein purification. Hands-on experience with performing experiments and analyzing data helps students to develop problem-solving skills that will benefit them throughout their life. It was suggested in a recent editorial in Science that 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 that have led to these explanations [1]. Laboratory courses, in particular, must move away from cookbook-type, observation-based experiments and offer a curriculum that helps to develop scientific reasoning [2, 3]. By allowing more complex questions to be presented, a project-based laboratory course can better achieve these problem-solving goals [4, 5]. This being said, it is often desirable to have multiple projects in the course so that the students can be exposed to different experimental systems and perform a wider variety of techniques. Projects that require too much time to complete can limit this, and force instructors to fill in the left over laboratory periods with less engaging, stand-alone experiments. Additional Supporting Information may be found in the online version of this article. To whom correspondence should be addressed. Tel.: ; abcoleman@ucsd.edu. This paper is available on line at The multistep purification of an enzyme from its source material, where students perform different purification techniques over successive laboratory days, naturally fits into a project-based biochemistry laboratory course. From a pedagogical perspective, an enzyme purification project serves two functions. First, it exposes students to a variety of techniques that are widely used in biochemistry. Second, each purification technique works by exploiting a different biochemical property of the enzyme so that a number of fundamental biochemical principles are demonstrated by the experiments. Lactate dehydrogenase (LDH) has long been a popular enzyme to work with in student laboratories [6, 7]. The starting material for its purification from mammalian tissue (bovine or porcine skeletal muscle, heart, and liver) is easy to obtain, and the enzyme is relatively stable in student hands. Different combinations of techniques, including size exclusion (SE), ion exchange, and affinity chromatography, can be used to purify the enzyme, giving the instructor the flexibility to design a purification strategy that covers the particular principles and techniques they want to teach [6]. The difficulty with this type of project is that a large number of laboratory periods are required to complete the procedures in a typical purification strategy, and the enzyme purification can quickly take over a laboratory DOI /bmb.20385

2 254 BAMBED, Vol. 38, No. 4, pp , 2010 course leaving little time for anything else. In particular, column chromatography steps typically require two laboratory days to complete, where students run the column on 1 day and then spend the next day screening fractions for enzyme activity. This can make much of the project repetitive and risks losing student interest. Other problems can include having a sufficient number of spectrophotometers that can perform kinetic measurements for activity assays, and the risk that the students will lose all of their enzyme activity during the purification. We set out to develop an enzyme purification laboratory project that would circumvent these difficulties, and fit into our Biochemical Techniques course in the Division of Biology at the University of California, San Diego. This is an independent, upper-division laboratory course that focuses on protein biochemistry techniques. The course has a separate lecture component and two 4-hour laboratories per week for the 10-week quarter. Most of the laboratory experiments are integrated into three modules that consist of an enzyme purification, a Western blot to determine inactivation of MAP kinase during sea urchin fertilization, and the expression/determination of unknown fluorescent proteins in bacterial cells. Biochemical technique is a high-enrollment course, with upward of 1,000 students completing the class each year when summer sessions are included. Therefore, sufficient availability of equipment and resources are an issue when implementing new experiments. In redesigning the enzyme purification module of the course, we sought to fulfill the following: The students would conduct each step of the purification and analysis, providing them with maximum investment in the project from the beginning to end. The project would be question-based, with an unknown that the students would have to answer. The project could be completed in a time frame that would not interfere with the other projects in the course. The equipment and facilities required, and the reagent costs involved, would be kept to a minimum. We decided on LDH as the target enzyme for our purification for the reasons mentioned earlier, as well as the fact that it is an intriguing enzyme for students to work with. LDH transfers electrons from NADH to convert pyruvate to lactic acid during low oxygen conditions in actively working muscle cells, replenishing the cytosolic supply of NAD þ and allowing for continued ATP production by glycolysis (Fig. 1) [8]. Lactate is later converted back to pyruvate by LDH in the liver, where it provides a carbon source for glucose synthesis. Glucose produced by the liver then replenishes the carbohydrate stores in the muscle cells, completing the Cori cycle. There are five different isozymes of LDH with distinct isoelectric points that can be separated electrophoretically [6, 9]. The isozymes derive from different combinations of muscle-type and heart-type subunits that come together in different combinations to make the active LDH tetramer. Tissue-specific expression of the subunits leads to production of distinct sets of isozymes in skeletal muscle versus heart tissue. Damage to cardiac tissue FIG. 1.Reaction catalyzed by lactate dehydrogenase. leads to an increase in the concentration of heart-type isozymes in serum, and LDH is one of the blood enzymes used to diagnose myocardial infarction [10]. We have used the tissue-specific expression of the different isozymes to add the identification of an unknown to the project. Porcine skeletal muscle and heart are prehomogenized for the students just before the first laboratory, and each laboratory group is given one or the other as an unknown crude homogenate. For the students, this frames the project from the beginning in terms of a question they will have to answer. To identify their unknown they must complete several problem-solving tasks, from calculating LDH activity units to analyzing the band positions on the agarose gel that is used to separate the isozymes. Our new enzyme purification project has accomplished the goals we had set for it. The project allows students to carry out an enzyme purification, which retains the pedagogic value of a long-term project, under circumstances where time and access to equipment are limited. Furthermore, the laboratories directly incorporate many items that have been identified by the American Society for Biochemistry and Molecular Biology as core content in undergraduate laboratory courses [11]. The combination of techniques used to purify the enzyme gives good recovery of LDH in just three laboratory sessions, while still achieving a high degree of purification. More importantly, the question-based analysis laboratories help to foster the problem solving skills that we hope to bring out in our students. LABORATORIES AND TEACHING POINTS Refer Table I for an overview of the project. The students in our Biochemical Techniques course work in laboratory groups of three, although groups of two students can easily complete all of the procedures. Ice is available, but the use of a cold room is not required for any of the procedures in the project. At the first lecture, we discuss the importance of keeping the purification intermediates as cold as possible to prevent degradation of LDH by proteases and suggest some strategies to help with this, such as prechilling glassware. During the purification phase of the project, the students save samples of the purification intermediates for analysis in laboratory session 4 (Table II). All chemicals were purchased from either Sigma Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA), unless otherwise indicated. For the purification laboratories (1 3), each procedure is carried out in 50 mm phosphate buffer at ph 7.5. A full description of the methods for each laboratory is available in the Supporting Information for this article.

3 TABLE I Laboratory schedule Laboratory session Procedures performed Approximate time required (hr) Laboratory 1 Initial purification of LDH from crude homogenate; 3 centrifugation and fractionation by ammonium sulfate precipitations Prepare columns for size exclusion chromatography Laboratory 2 Affinity chromatography purification of LDH 4 Screen chromatography fractions Concentrate combined fractions Laboratory 3 Size exclusion chromatography purification of LDH 3.5 Screen chromatography fractions Concentrate combined fractions Laboratory 4 Kinetic LDH activity assays 3.5 Bradford protein assays Laboratory 5 Agarose gel electrophoresis to determine LDH isozymes 3 The project can be completed in five 4-hr laboratory days. The approximate time required for students to complete each laboratory is based on our observations of the time it takes for greater than 90% of the laboratory groups to finish all the procedure for that day. 255 Laboratory Session 1 The students receive an unknown crude homogenate of either porcine heart or skeletal muscle. The crude homogenate is clarified with an initial centrifugation, and then further fractionated by ammonium sulfate precipitation (a 40% cut followed by a 70% cut) [6]. The homogenization of the heart and skeletal muscle tissue is the only step of the purification that is not performed by the students. This saves time by bypassing a step where students frequently get backed up, and allows the element of an unknown to be added to the project. A sample of the supernatant from the initial centrifugation (referred to as the clarified homogenate) and a sample of the resuspended 70% cut pellet are retained for later analysis. A sample of the original crude homogenate can also be saved, but this fraction is difficult to analyze because of its turbidity and the susceptibility of the LDH to proteolytic degradation. Dialysis is often performed on the prep at this point to remove residual ammonium sulfate that could potentially inhibit binding of LDH to the affinity column in the next laboratory. We have found that any residual ammonium sulfate present in the resuspended 70% pellet has no affect on LDH binding to the Cibacron Blue affinity resin. As performing the dialysis procedure frequently causes students to lose much of their LDH activity, this step was left out of the purification scheme. For the first lecture of the project, the idea of tailoring a purification strategy to target a particular enzyme is presented to the students. We emphasize the importance of knowing the cellular localization of the enzyme, and discuss how the initial centrifugation they perform isolates the cytosolic fraction of the cell where LDH resides, and removes organelles like the nucleus and lysosomes. As part of their final laboratory report for the project, the students are required to find an article from the literature that describes the purification of another enzyme, and compare and contrast the purification strategy used for that enzyme to the strategy they used to purify LDH. The concept of protein hydrophobicity is also discussed to explain how the ammonium sulfate precipitations are used to separate proteins based on differences in their solubility. Laboratory Sessions 2 and 3 The students continue the LDH purification with two column chromatography steps. Affinity chromatography with Cibacron Blue 3GA-agarose is performed in laboratory session 3, followed by size exclusion chromatography in laboratory session 4 to yield their final, purified LDH. During the affinity chromatography run, the students track elution volume (V e ) and monitor total protein elution from the column with periodic absorbance measurements at 280 nm (A 280 ). Fractions of the column output are collected for both the affinity and size exclusion chromatography purification steps. Testing each fraction with a standard kinetic assay for LDH would require additional laboratory sessions and a large number of spectrophotometers capable of performing kinetic measurements. Therefore, we devised a qualitative, colorimetric assay for LDH that TABLE II Student purification table Purification step LDH activity units (U) Total protein (mg) Specific activity (U/mg) Percent recovery Fold-purification Clarified homogenate % pellet Affinity-purified LDH SE-purified LDH The data shown was generated by a single student laboratory group during Winter Quarter of Column 1 lists each purification intermediate and the final SE-purified LDH. The purification was done from skeletal muscle, and the SE-purified LDH was concentrated by ammonium sulfate precipitation. Samples from each step were analyzed to determine the total LDH activity units present (column 2) and the amount of total protein present, as determined by Bradford assay (column 3). Percent recovery is the percent LDH activity units remaining at each step, relative to the clarified homogenate. Fold-purification is the ratio of the specific activity from each step over the specific activity of the clarified homogenate.

4 256 BAMBED, Vol. 38, No. 4, pp , 2010 allows students to quickly analyze their fractions the same day that they run the column. The assay is based on the method of Allen for cytochemical visualization of LDH isozymes in electrophoresis gels [12]. Creation of NADH in the presence of LDH drives the successive reduction of phenazine methosulfate and then nitroblue tetrazolium (NBT). The reduced form of NBT absorbs strongly at 610 nm, producing a blue color. By setting up the assay components in a 96-well plate, it can be used to rapidly test large numbers of samples for LDH activity without the need for a spectrophotometer or plate reader. We refer to this LDH screening assay as the spot test. The appearance of strong blue in a well after incubating 5 min indicates the presence of a significant amount of LDH activity in the corresponding fraction (see Supporting Information for a full description of the assay). The Cibacron Blue moiety on the affinity resin mimics the structure of adenosine nucleotides, and fits into the nucleotide-binding pocket of enzymes with nucleotide cofactors [13]. It is frequently used for the purification of enzymes that utilize NAD þ and ATP as substrates [14]. In lecture, the structure of the NAD þ binding site on an LDH subunit is examined, providing an example of how a substrate interacts with the active site of its enzyme. This opportunity is also taken to introduce the concept of affinity interactions between biological molecules and the quantification of their binding strength by dissociation constant. Although this subject is seldom emphasized in undergraduate coursework, high-specificity, high-affinity interactions between biomolecules are fundamental to all fields of biochemistry and cell biology, and the binding of an enzyme to a substrate analog on an affinity resin provides an excellent platform to introduce this. The A 280 measurements of the column output allow the students to visualize protein binding and elution during the chromatography run. The majority of the proteins in the second peak that elute with 1 M NaCl are nucleotide binding proteins (Fig. 2). For their final laboratory report, the students prepare a plot of V e versus A 280, giving them the equivalent of the UV trace that would be recorded from a more sophisticated column setup with a UV detector. They then superimpose their fraction numbers at the corresponding V e on the x-axis of the UV trace, and indicate which fractions were positive for LDH activity. This allows them to see specifically where LDH eluted in relation to the other proteins present in the 70% pellet. Laboratory Session 4 The students now perform kinetic assays for LDH activity and Bradford protein assays, which allow them to assess the number of LDH activity units and the total protein content at each step in their purification strategy. The standard LDH activity assay measures NADH production over time by tracking the change in absorbance at 340 nm, as described previously [6]. The extinction coefficient for NADH is 6220 cm 21 M 21 at 340 nm, and one LDH activity unit is defined as 1 lmol NADH/min (1 U). Although the activity assays can be performed on a basic UV spectrophotometer by having students take FIG. 2. Column chromatography steps and analysis of fractions by the spot test for LDH activity. The data shown are from a single, representative student laboratory group. (a) Protein elution during the affinity chromatography with Cibacron Blue-agarose. Elution volume begins at the time the sample was added to the column (V e ¼ 0), and absorbance at 280 nm was measured every 2 3 ml following addition of the sample (filled diamonds). 1 M NaCl was added to elute bound proteins at V e ¼ 29 ml and 1 ml fractions were collected from this point (shown at corresponding V e ). (b) Spot test to detect LDH activity in the fractions collected from the affinity chromatography run shown in (a). One hundred microliters of master mix was added per well in a 96-well plate, and 2 ll of the indicated fraction (1 20) was added to each well. Two microliters of 70% pellet was added to the positive control well (þ), and nothing was added to the negative control well (2). (c) Spot test to detect LDH activity in fractions collected from the size exclusion chromatograph with Sephadex G100. The assay was performed as in (b). timed absorbance readings, more reliable results are obtained on a spectrophotometer that has a programmable kinetics function. By having half the class begin the Bradford protein assays first, we find that one spectrophotometer for every six laboratory groups gives a reasonable amount of access to the instruments. The students need some coaching on how to perform the steps of the assay quickly enough to catch the beginning of the reaction. The importance of quantifying enzymes by enzyme activity units is discussed in the lecture. The students then learn how to interpret their kinetic data and calculate LDH activity units [6]. Particular attention is paid to making sure that their absorbance versus time plot is linear so that they are using the initial reaction velocity for their calculations. After determining the change in absorbance (DA/min) for an assay, we cover each step of the calcula-

5 257 tions to determine relative activity (U/mL), total activity (U), percent recovery, total protein concentration (mg/ml), total protein (mg), specific activity (U/mg total protein), and fold purification. This culminates in the enzyme purification table that the students prepare for their laboratory report. Table II lists the aforementioned values for each step in the purification process, and lets the students track the removal of contaminating proteins and the recovery of LDH with each step. In the laboratory report, they are required to critique their purification strategy, and identify the most effective and least effective purification steps. The effectiveness of any step is based on both the recovery of LDH and how well the procedure worked to remove contaminating, non-ldh proteins. Finally, based on what they know about how each technique works, and what they have learned about other purification techniques in the lecture, they are asked to propose a change that will improve their purification strategy. This approach gets the students to look at their data with a much more critical eye. It also ensures that they understand what each value in their purification table represents; for instance, that milligrams of total protein does not mean milligrams of LDH, or that they can have an increase in protein concentration and still have a lower total protein value if that purification step results in a much smaller volume. By having to answer a specific set of questions, the students become much more engaged in thinking about how each value changes over the course of the purification. Laboratory Session 5 In the final laboratory of the project, the students analyze samples of their SE-purified LDH by agarose gel electrophoresis. An LDH-specific activity stain is used to visualize the isozyme bands in the gel [6]. This is a classic, relatively simple experiment for the teaching laboratory that can be done with either agarose or polyacrylamide gels [15], and here we have set up the experiment so that the students can identify the unknown isozymes in their purified LDH. The students run 1 U of their LDH on the gel along with heart and muscle-type isozyme standards, and they are required to calculate the volume of SE-purified LDH that contains 1 U. The identification of the LDH isozymes based on their electrophoretic migration provides an excellent platform to present the biochemical properties of proteins that determine their net charge at any given ph. The ability of ph to alter the charge on the side chains of individual acidic and basic amino acids, how this affects the net charge of the protein, and the concept of the isoelectric point (pi) are discussed in some detail. The subunit composition of the different LDH isozymes is also covered in more detail at this point. Tissue-specific expression of the muscle-type (M) and heart-type (H) subunits produces M 4 and M 3 H tetramers in skeletal muscle that are the muscle isozymes. Cardiac muscle, on the other hand, produces H 4 and H 3 M tetramers that are the heart isozymes. Beyond merely demonstrating these concepts, the isozyme determination experiment is designed to get as much independent thinking out of the students as possible. The students are not given the identity of the isozyme standards. They are required to do a brief bioinformatics analysis to determine the pi of the muscle and heart isozymes. This allows them to figure out which isozymes are expected to migrate more quickly in the gel, and thus identify their unknown. They are given the necessary accession numbers, and must first look up the amino acid sequences of the porcine M and H subunits in the NCBI protein sequence database [16]. They then use the ProtParam tool at the ExPASy proteomics website to analyze both amino acid sequences [17]. The ProtParam tool uses different programs to calculate or estimate of some of the protein s biochemical properties, including pi, based on the input sequence. It also gives the numbers of the different charged amino acids in the M and H subunits, which aids in the student s understanding of where the difference in pi comes from. The porcine M and H-type LDH subunits have respective NCBI accession numbers of AAA50436 and AAA50438, and their respective pi values are 8.18 and The students then estimate the pi of the muscle and heart isozyme tetramers by taking the average pi value for the four subunits composing that isozyme (e.g. the M 3 H isozyme has a pi of 7.53). On the basis of the pi values of the isozymes, they deduce the muscle and heart isozyme standards from their relative band positions in the gel. By comparing the band position of their unknown, SE-purified LDH to the standards, the students can determine which isozymes they have purified, and what was there as starting material. RESULTS AND DISCUSSION We have been using the LDH purification and analysis project in our Biochemical Techniques course for 1 year now, and it has been taught in different course sections by four instructors. The design of the affinity chromatography laboratory (laboratory session 2) has worked particularly well to engage the students in what can otherwise be a long and tedious experiment. By tracking protein output from the column with periodic A 280 measurements, they play an active role in deciding how long to run the washing and elution steps of the chromatography. Constructing the plot of A 280 versus V e then gives them a record of their chromatography run. For the plot shown in Fig. 2a, the sample was loaded on the column over the first 5 ml of V e and was washed with phosphate buffer to a V e of 29 ml. At this point, 1 M NaCl was added to elute the bound proteins. The plot clearly shows distinct peaks for the unbound proteins that come out in the column wash and the bound proteins that elute with 1 M NaCl. By virtue of the Cibacron Blue resin, the bound proteins should consist mainly of NAD þ and ATP-binding proteins. The number of each 1 ml fraction is shown at its corresponding V e (Fig. 2a), and the students are required to indicate this on the plots they create for their laboratory reports.

6 258 BAMBED, Vol. 38, No. 4, pp , 2010 LDH activity units (U) TABLE III Statistical analysis of LDH recovery from student laboratory groups Concentrated SE-purified LDH Nonconcentrated SE-purified LDH Total protein (mg) Specific activity (U/mg) LDH activity units (U) Total protein (mg) Specific activity (U/mg) n 20.* ** 21.* ** Mean Median Range Statistical analysis was done for quantity and purity of SE-purified LDH recovered by student laboratory groups during winter quarter Data are shown from a set of student groups that performed a final ammonium sulfate precipitation to concentrate their SE-purified LDH, and a separate set that did not concentrate the SE-purified LDH. Both sets were evenly divided between heart and skeletal muscle as the starting tissue for the purification. The single highest and lowest values were eliminated from the analysis for each column, and laboratory groups that were unable to generate data with a particular assay were also not included (not >3 for any column). n is the number of student groups used to calculate the statistical values in each column. * By Student s t-test, p ¼ ** By Student s t-test, p ¼ The students take their decision making role in the experiment further when they decide which fractions to combine based on the results of the spot test for LDH activity. The spot test shown in Fig. 2b is from the fractions collected during the affinity column run shown in Fig. 2a. The test shows no color change for fractions 1 4, an intense blue color from fractions 5 10, and a gradual decrease in the amount of blue color from fractions Weak blue color can be produced from as little seven LDH units in a 1 ml fraction ( U/2 ll added to the spot test well), so most of the LDH activity was retrieved from the column before fraction 20. The student group that conducted this experiment kept fractions 5 15 for their affinity-purified LDH. The spot tests of some laboratory groups show strong blue color through fraction 20, indicating that some LDH remains on the column, even though most of these groups also show that A 280 has returned to baseline by this time. We have students stop collecting at fraction 20 to prevent the laboratory from running overtime; however, in some cases collecting more fractions might increase the recovery of LDH. The fractionation range of the Sephadex G100 resin used for the size exclusion chromatography is kda. The LDH tetramer is kda, and thus is just below the exclusion limit for the resin. This allows for efficient removal of lower-mw proteins, but causes the LDH to elute with the Blue Dextran in the first fractions. The Blue Dextran is diluted out when a sample of the fraction is added to the spot test and does not pose a problem in reading the assay. The students begin collecting fractions when the Blue Dextran is 1 cm from the bottom of the column, so both the tracking dye and LDH are present in fraction 1. Figure 2c shows a typical spot test result from the size exclusion chromatography run of a student laboratory group, where LDH is usually contained in the first eight fractions (the Blue Dextran usually elutes in the first three to four fractions). The students again play an active role in the experiment by deciding how many fractions they will keep. They understand that by excluding the fractions that produced lighter blue color they may slightly lower their yield, but can increase their degree of purification by removing more contaminating proteins. For the spot test shown in Fig. 2c, the students kept fractions 1 3. The overall purification strategy that we have described allows almost all of our student laboratory groups to successfully purify a good quantity of LDH. The LDH recovery of our student laboratory groups from winter quarter of 2009 was statistically examined (Table III). The median quantity of LDH recovered from 25 g of starting tissue was 340 U from 20 student groups who concentrated their SE-purified LDH with a final ammonium sulfate precipitation, and 134 U from 21 student groups who did not concentrate their SE-purified LDH. The laboratory groups in both sets were evenly divided between heart and skeletal muscle as the starting tissue. The higher yields obtained with concentrating the LDH may result from greater stability of the enzyme when stored this way, and we now include the final ammonium sulfate precipitation in our student procedure. The only disadvantage to performing the final ammonium sulfate precipitation is that the total protein concentration of the SE-purified LDH is low, and occasionally a laboratory group will have difficulty getting their material to precipitate. Data from an instructor s test run of the purification is available in the Supporting Information for comparison. The SE-purified LDH produced by the students has a relatively high degree of purity. SDS-PAGE analysis shows a single prominent band corresponding to LDH with some faint background bands (data not shown). Table II shows a representative student purification table. This data set was chosen from the winter 2009 class by selecting the laboratory group with the LDH final yield that was closest to the class average of 497 U (concentrated SE-purified LDH). The students do not analyze samples of the original crude homogenate from the beginning of the purification, and for the purposes of the purification table the starting point is considered the clarified homogenate. The initial centrifugation to produce the clarified homogenate removes most of the cellular organelles and leaves primarily the cytosolic fraction. Thus, to give the students cleaner data to interpret, the purification table begins from an already partially purified intermediate. A big part of the analytical work the students do at the end of the project is in examining their purification tables to critique the purification strategy they used to purify LDH. The most and least-effective purification steps can vary between laboratory groups, and there is no set right

7 259 FIG. 3.Identification of LDH isozymes by agarose gel electrophoresis. Student unknown LDH samples and isozyme standards were run on 1% agarose gels at ph 9.5 at 80 V. For both gels, 1 U of the unknown, SE-purified LDH (student LDH) was run, along with 4 U each of LDH from rabbit skeletal muscle (muscle standard) and bovine heart (heart standard). (a) Student LDH was purified from porcine heart. (b) Student LDH was purified from porcine skeletal muscle. or wrong answer as long the students effectively use the values in their purification table to justify their decisions. Typically, and not unexpectedly due to the specific nature of the interaction, the affinity chromatography step proves to be most effective as determined by the decrease in total protein and the increase in fold-purification (Table II). However, depending on how successful a student group is in recovering their LDH from the affinity column, their purification table may show that other steps were more effective in their particular prep. The identification of the unknown isozymes in the last laboratory provides an exciting climax to the project. Comparison of the bands from the students SE-purified LDH with bands from the standards usually allows for easy identification of the unknown isozymes. Figure 3 shows two gels that were run by student laboratory groups. The unknown sample (student LDH) for Fig. 3a was purified from heart tissue, and for Fig. 3b was purified from skeletal muscle. The skeletal muscle isozymes carry less negative charge than the heart isozymes at the gel ph of 9.5 and migrate more slowly in the gel. The heart isozyme standard is from bovine heart (porcine is not available), and the species difference seems to cause a slight difference in migration between the heart standard and the porcine heart LDH that the students purify (Fig. 3a, compare heart standard with student LDH). Also, the porcine skeletal muscle frequently shows small amounts of heart-type isozymes in addition to the more prominent skeletal muscle isozymes, and this is seen as the less intense, more quickly migrating band in the student LDH lane of Fig. 3b. It is known that skeletal muscle expresses small amounts of the heart-type isozymes [18]. Neither of these issues, however, seem to pose a problem for the correct identification of the unknown isozymes. The LDH purification project has proved to be a successful addition to our Biochemical Techniques course. Nearly all of the students are able to produce quality data and correctly complete the required analysis. The student response to the project has also been positive. When a recent class was surveyed, almost all the students responded that the project facilitated their learning about protein purification techniques and that they were interested or engaged in the project (see Supporting Information for the complete survey). We also believe the project has enhanced the degree to which the course curriculum challenges the students to use scientific reasoning. The project incorporates many elements that have been suggested by Harwood to promote active learning in laboratory classes, such as getting away from simply verifying known results, and getting students to look at the scientific literature [19]. Although the laboratories are structured enough to carry out in a high-enrollment course, they retain the problemsolving theme that promotes learning [2]. Student feedback supports these conclusions. The survey asked their level of agreement with the statement, analyzing your data for the experiments in the LDH purification project required you to use scientific reasoning, and 82.4% of the class either agreed or strongly agreed with the statement, 14.7% moderately agreed, 2% were uncertain, and 0% disagreed. Furthermore, they were asked compared to experiments you have done in other chemistry and biology laboratory courses you have taken, how

8 260 BAMBED, Vol. 38, No. 4, pp , 2010 much did the experiments and data analysis you performed in the LDH purification project help to promote the development of problem-solving skills? 31.8% of the students responded more than in other laboratory courses, 24.2% responded slightly more than in other laboratory courses, 31.8% responded the same as in other laboratory courses, and 12.1% responded less than in other laboratory courses. The LDH that the students purify would be suitable for other applications in a biochemistry laboratory course, both in quantity and degree of purity. The purification and analysis project we have described here could easily be done as a module of a larger project. In particular, LDH is commonly used to study enzyme kinetics, and several good sets of experiments have been published on this [6, 7]. We have found this project to be enjoyable for the students to perform and for the faculty to teach. By promoting question-based thinking in our students while they conduct experiments that demonstrate many of the fundamental concepts in biochemistry, we believe we are achieving the best possible outcome from our Biochemical Techniques course. REFERENCES [1] B. Alberts (2009) Redefining science education, Science 323, 437. [2] C. Gallet (1998) Problem-solving teaching in the chemistry laboratory: Leaving the cooks, J. Chem. Educ. 75, [3] J. Handelsman, D. Ebert-May, R. Beichner, P. Bruns, A. Chang, R. DeHaan, J. Gentile, S. Lauffer, J. Stewart, S. M. Tilghman, W. B. Wood (2004) Scientific teaching, Science 304, [4] P. A. Craig (1999) A project-oriented biochemistry laboratory course, J. Chem. Educ. 76, [5] G. W. Muth, J. W. Chihade (2008) A streamlined molecular biology module for undergraduate biochemistry labs, Biochem. Mol. Biol. Educ. 36, [6] S. O. Farrell, R. T. Ranallo (2000) Experiments in Biochemistry: A Hands-on Approach, Brooks/Cole, Florence, KY. [7] J. L. Powers, N. E. Kiesman, C. M. Tran, J. H. Brown, V. L. H. Bevilacqua (2007) Lactate dehydrogenase kinetics and inhibition using a microplate reader, Biochem. Mol. Biol. Educ. 35, [8] D. L. Nelson, M. M. Cox (2008) Lehninger: Principles of Biochemistry, 5th ed., W. H. Freeman, New York. [9] G. S. Zhuang, J. Liu, C. P. Jia, Q. H. Jin, J. L. Zhao, H. M. Wang (2007) Microchip-based capillary electrophoresis for determination of lactate dehydrogenase isoenzymes, J. Sep. Sci. 30, [10] J. A. Lott, J. M. Stang (1980) Serum enzymes and isoenzymes in the diagnosis and differential diagnosis of myocardial ischemia and necrosis, Clin. Chem. 26, [11] J. G. Voet, E. Bell, R. Boyer, J. Boyle, M. O Leary, J. K. Zimmerman (2003) Recommended curriculum for a program in biochemistry and molecular biology, Biochem. Mol. Biol. Educ. 31, [12] J. M. Allen (1961) Multiple forms of lactate dehydrogenase in tissues of the mouse: Their specificity, cellular localization, and response to altered physiological conditions, Ann. N Y Acad. Sci. 94, [13] J. F. Biellmann, J. P. Samama, C. I. Bränden, H. Eklund (1979) X-ray studies of the binding of cibacron blue F3GA to liver alcohol dehydrogenase, Eur. J. Biochem. 102, [14] V. Bohácová, P. Docolomanskỳ, A. Breier, P. Gemeiner, A. Ziegelhöffer (1998) Interaction of lactate dehydrogenase with anthraquinone dyes: Characterization of ligands for dye-ligand chromatography, J. Chromatogr. B Biomed. Sci. Appl. 715, [15] G. P. Manchenko (2002) Handbook of Detection of Enzymes on Electrophoretic Gels, 2nd ed., CRC Press, Boca Raton, FL. [16] National Center for Biotechnology Information (NCBI), Available at: Accessed on July 1, [17] ExPASy Proteomics Server of the Swiss Institute of Bioinformatics, Available at: Accessed on July 1, [18] G. van Hall (2000) Lactate as a fuel for mitochondrial respiration, Acta Physiol. Scand. 168, [19] W. S. Harwood (2003) Course enhancement: A road map for devising active-learning and inquiry-based science courses, Int. J. Dev. Biol. 47,