Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX
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Using a Simple, Inexpensive Undergraduate Isoelectric Focusing Experiment for Proteins and Nanomolecules To Help Students Understand Isoelectric Point and Its Real-World Applications A. Sharma,* H. Kopkau, and D. Swanson Department of Chemistry & Biochemistry, Central Michigan University, Mt. Pleasant, Michigan 48859, United States
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S Supporting Information *
ABSTRACT: A laboratory experiment was designed for upper-level undergraduate students in a bioanalytical course to demonstrate the concept of isoelectric point (pI) and the theoretical and practical applications of isoelectric focusing (IEF). This simple and inexpensive IEF procedure requires the same minigel SDS-PAGE equipment that is already available in most biochemistry and biology laboratories. Students first use published mathematical formulas to predict the pI of a nanomolecule known as a polyamidoamine dendrimer and subsequently determine its pI by preparing and running an IEF gel using common proteins with well-established pI values as standards. The pI of the nanomolecule is determined from a calibration plot generated between migration distances and pIs of the protein standards. This experiment has been successfully performed by over 50 bioanalytical students over the past five years. Evidence from in-lab problems, lab reports, and postlab quizzes shows that the use of medical examples, protein computer databases for determination of pI from amino acid composition, and determination of the pI from an IEF gel help students understand the difficult concept of isoelectric point and appreciate its applications in the real world. KEYWORDS: Upper-Division Undergraduates, Biochemistry, Hands-On Learning/Manipulatives, Bioanalytical chemistry, Electrophoresis
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INTRODUCTION Isoelectric focusing is often the first dimension of twodimensional electrophoresis, which is a powerful method for separating a large number of proteins present in a mixture such as cell lysates and human fluids. The second dimension is typically SDS-PAGE. While SDS-PAGE is a popular experiment in undergraduate biochemistry laboratories, IEF is not often taught.1,2 One of the major reasons for this is the expense of the equipment and supplies involved in teaching an IEF laboratory experiment, especially when using immobilized pH gradient (IPG) strips. Undergraduate students in biochemistry and biology often have difficulties in understanding the concept of isoelectric point of a biomolecule, which requires knowledge from general chemistry (pKa) as well as organic chemistry (functional groups that act as weak acids). Determining the pI values of large proteins or macromolecules is complex and often requires the use of computer programs.3−5 In addition, biochemistry lectures typically do not stress the biomedical applications of isoelectric points although many students in a typical biochemistry class are pursuing biological or biomedical careers. Isoelectric points also play an important role in the area of nanotechnology where large macromolecules are designed and synthesized to accomplish specific biological functions. A popular nanomolecule that has been extensively studied is the polyamidoamine (PAMAM) dendrimer.6,7 PAMAM den© XXXX American Chemical Society and Division of Chemical Education, Inc.
drimers are synthetic, branched macromolecules that span a size range similar to that for typical proteins. They are soluble in water and are commercially available in a variety of sizes and surface chemistries. A PAMAM dendrimer molecule consists of a core, cavities, and surface functional groups. The interior of the dendrimer consists of an amide backbone with branches emanating from tertiary amines. A generation 2 (G2) dendrimer (MW ∼ 3300 Da) with a cystamine core, 14 tertiary amines within the interior of the macromolecule, and a surface with 16 primary amine groups (the number of interior tertiary amines is two less than the number of surface groups) is shown in Figure 1. Other commonly used PAMAM dendrimers include generation 4 (G4) with a diaminobutane (DAB) core, 62 tertiary amines, and surfaces composed of 64 amine groups (known as G4 DAB amine-surface dendrimer), 64 carboxyl groups (carboxyl-surface dendrimer), or 64 hydroxyl groups (hydroxyl-surface dendrimer). Dendrimers with mixed surfaces are also available. For example, a G4 DAB 90/10 contains 90% hydroxyl groups (58 −OH) and 10% amines (6 −NH2). Typical pKa values for these groups have been reported to be ∼9 for the surface primary amines, ∼6 for the interior tertiary amines, and ∼15 for the surface hydroxyls. Received: April 17, 2018 Revised: June 26, 2018
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DOI: 10.1021/acs.jchemed.8b00184 J. Chem. Educ. XXXX, XXX, XXX−XXX
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characterize an unknown protein and a nanomolecule using various bioanalytical techniques and protein databases. Gel Preparation
Students first prepare a 7% polyacrylamide gel by mixing water, acrylamide/bis-acrylamide, urea, and carrier ampholytes. The gel takes about 45−60 min to polymerize. While students wait for the gel to polymerize, a prelaboratory lecture is given. Details of the prelaboratory material, gel preparation, and the IEF procedure are given in the Supporting Information. Electrophoresis
Any SDS-PAGE equipment (minigel system) may be used for the IEF experiment. The inner buffer chamber (facing the samples) is filled with anolyte solution (phosphoric acid) while the outer chamber is filled with catholyte solution (NaOH). Samples are mixed with equal volumes of an IEF sample buffer and applied into the gel lanes. Bromophenol blue and detergents are omitted from the sample buffer due to their possible interactions with the dendrimer. The detergent is required if running poorly soluble proteins (such as membrane proteins). All of the proteins and the dendrimer used in this experiment (including the unknown hen egg white lysozyme) are water-soluble. The gel is run for 160 V (initial current is about 11 mA) until the current reaches 3 mA. The voltage is then increased to 300 V (maximum voltage with our power supply) to keep the current above 3 mA. It is important to remind students to reverse the electrical connections to the power supply (the red and black wires from the chamber are connected to the black and red inputs of the power supply, respectively) so that the bottom of the gel is the cathode (negative electrode in electrophoresis). When the current gets too low the power supply automatically stops. The run typically takes 60−90 min. In the case of time constraints, 1 h at maximum voltage may be used; students obtain good results when the cytochrome c migrated more than halfway down the gel. After electrophoresis, the gel is rinsed with deionized water and then washed in methanol/acetic acid destaining solution. This step is important since it removes any carrier ampholytes, which interfere with gel staining. Excessive washing should be avoided since dendrimers, unlike proteins, do not denature and become fixed and may therefore diffuse out of the gel. This is the reason a high generation dendrimer with a molecular weight over 10 kDa is used. Although smaller dendrimers are cheaper to buy, they tend to diffuse more readily than larger ones. As with SDS-PAGE, staining is carried out with coomassie blue and destaining with methanol/acetic acid.
Figure 1. A Generation 2 PAMAM dendrimer with a cystamine core, tertiary amines (red), and amine surface groups (blue).
A generation 4 PAMAM dendrimer has a molecular weight of ∼15,000 Da. Unlike proteins, PAMAM dendrimers do not selfaggregate and can be frozen for storage and thawed before use for many years. This is convenient for teaching laboratories since their solutions may be made in large quantities and stored frozen before and after use. In the proposed laboratory experiment, students use isoelectric focusing to determine the isoelectric points of proteins and nanomolecules and compare their experimentally measured pI values with predicted pI values from mathematical formulas. The proposed IEF protocol requires the same electrophoresis equipment used for SDS-PAGE, which is typically available in most undergraduate teaching laboratories.8,9 The laboratory experiment involves the following: • Use of mathematical formulas for predicting the isoelectric points of standard proteins (cytochrome c, ribonuclease A, myoglobin, amyloglucosidase) and a nanomolecule (PAMAM dendrimer) • Running protein standards, an unknown protein, and a nanomolecule (G4 DAB 90/10) on an IEF gel • Plotting a calibration curve • Calculating the pI of the unknown protein and nanomolecule After completion of this laboratory exercise students should be able to • Relate the pI of proteins and nanomolecules to the number and type of functional groups present in the macromolecules • Explain the use of pI in real-world applications such as why proteins in the bloodstream typically have pI values less than 9; the importance of high pI values to the functions of basic proteins such as myelin basic protein, lysozyme, cytochrome c, histones, and protamines; and the preparation of short-acting, intermediate-acting, and long-acting insulin (discussed in Supporting Information)
Predicting pI Values of Proteins and Dendrimer
While waiting for electrophoresis, students calculate the pI of a tripeptide given its pKa values. They predict the isoelectric points for standard proteins (cytochrome c, ribonuclease A, myoglobin, and amyloglucosidase) and a nanomolecule (G4 90/10 dendrimer) using mathematical formulas that are based on the number of acidic and basic amino acids, terminal amine, and carboxyl groups as well as tyrosine and histidine residues in a given protein.4 They are also given the pI values of the standard proteins published by the vendor. Using a similar rationale, students calculate the pI of the G4 90/10 dendrimer.
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HAZARDS AND WASTE DISPOSAL Chemical hazards and precautions for the laboratory experiment are given under Supporting Information. Excess chemicals such as ammonium persulfate or used solutions
EXPERIMENTAL DETAILS Students work in pairs to complete the lab within 4 h. This laboratory experiment has been performed for five years with 50 students (about 10 students per year) as part of a project to B
DOI: 10.1021/acs.jchemed.8b00184 J. Chem. Educ. XXXX, XXX, XXX−XXX
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prevents their diffusion during the staining and destaining step thereby giving sharp bands on electrophoresis gels. Dendrimers are not fixed and may give more diffuse bands, an effect which becomes worse with decreasing molecular weights. Working with dendrimers also exposes students to the world of nanotechnology. Students are fascinated to learn that the cavities of dendrimers may be loaded with drugs and their surfaces decorated with targeting ligands and imaging chemicals for selectively sending the drug payload to specific cells in the body that have receptors for the targeting ligand. The imaging agent on the dendrimer allows all of this to be visualized at the same time!12 Targeted therapy is becoming an important field in medicine.13 A representative gel obtained by students is shown in Figure 2. Under these conditions, the red cytochrome c band (lane 6) travels almost two-thirds down the gel.
(staining/destaining solutions and unpolymerized gel solutions) should be discarded into appropriate waste containers. After a picture of the gel is taken, the gel is discarded into a hazardous waste container. Due to the high voltage used in electrophoresis, students follow all prudent electrical safety rules.
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RESULTS AND DISCUSSION Students first determine the pI of a simple tripeptide, such as Lys-Phe-Asp, given the pKa values of the N- and C-terminals and the side chains of Lys and Asp. Most students remember how to calculate the pI value of an amino acid with two pKa values but either have forgotten or do not know how to calculate the isoelectric point of a chemical with more than two pKa values. This problem helps students better appreciate the next step, which is to use mathematical formulas for calculating the pIs of standard proteins without a computer.4 In order to do this, students have to first determine the amino acid composition of the protein and the number of lysine (K), arginine (R), histidine (H), aspartic acid (D), glutamic acid (E), cysteine (C), and tyrosine (Y) residues in the protein. This is done as follows: (1) Students obtain the UniProtKB identifier number of the protein by typing the name of the protein in the UniProt database.10 For example, for bovine cytochrome c, the number is P62894. (2) The number is entered into the Protein Information Resource Composition Database11 to obtain the amino acid composition of the protein. (3) Students then use the mathematical formulas4 to calculate the isoelectric points of cytochrome c, ribonuclease A, myoglobin, amyloglucosidase, and G4 90/10 dendrimer. Worked examples on calculating the pI values of ribonuclease A and the dendrimer are in the Supporting Information. This experiment helps students appreciate the effect of the amino acid composition on its isoelectric point and also become familiar with the use of protein databases. Students were successful in predicting the pI values of cytochrome c and ribonuclease A, but the mathematical formulas did not work for myoglobin, which has the same number of acidic amino acids (D and E) and basic amino acids (K and R). For amyloglucosidase (Aspergillus niger), students could not calculate its pI because the table of predicted pI values4 does not go below pI 4.0. Comparing proteins with the dendrimer also helps students better appreciate the nature of a protein such as the unfolding of its polypeptide chain with buried hydrophobic groups, the ability of unfolded chains to aggregate in organic solvents when their hydrophobic groups are exposed to the surface, and a protein’s compact interior region free of any cavities. On the other hand, a PAMAM dendrimer is a branched macromolecule rather than a folded chain. Due to its branched backbone, the interior of the dendrimer is rich in cavities. In addition, dendrimers do not have hydrophobic residues that are found in proteins. Due to the lack of folding and absence of nonpolar groups, dendrimers do not aggregate in organic solvents. Dendrimers are often stored in methanol. These differences are important for students to understand since dendrimers, unlike proteins, are not “fixed” during staining and destaining by methanol/acetic acid solutions. Many students do not appreciate the importance of the fixing step in electrophoresis. Proteins unfold and aggregate, which
Figure 2. Example IEF gel obtained by students: lanes 1 and 2 are the unknown protein (hen egg white lysozyme), lane 3 is amyloglucosidase (pI 3.6), lane 4 is myoglobin (pI 7), lane 5 is ribonuclease A (pI 9.6), lane 6 is cytochrome c (pI 10.5), and lanes 7 and 8 are the nanomolecule (G4 90/10 dendrimer).
The electrophoresis run is conveniently monitored by the migration of the cytochrome c sample (pI 10.5) toward the bottom of the gel. At the other extreme, amyloglucosidase, which has a low pI (3.6), remains very close to the top of the gel (lane 3). Although the amyloglucosidase band is not visible in ∼30% student gels, the other three proteins were sufficient to generate a linear calibration plot. Students are reminded to monitor their current during electrophoresis. Over 90% students have the habit of monitoring the voltage, which does not change under fixed-voltage runs and therefore does not provide useful information during electrophoresis. A rapid drop in current (within 5−10 min) is often due to leaks. Under normal circumstances, the current should slowly drop to around 3−4 mA in 60−90 min. If cytochrome c is only halfway down the gel, the voltage is increased to maximum. This will maintain the current at the 3−4 mA range to allow for further migration of cytochrome c. Unlike SDS-PAGE, students are reminded that the proteins cannot runoff the gel as they will stop migrating when they reach their pI values. The IEF could also be run by adding the catholyte solution (NaOH) to the upper (inner) buffer chamber. In this case, the red and black wires will not be switched (red to red and black to black). However, cytochrome c will remain close to the top of the gel and will not be helpful in visually monitoring the electrophoresis run. Washing the gel with destaining solution to remove the ampholytes after electrophoresis and before staining is an important step. The washing time for destaining was optimized to remove the ampholytes without causing significant distortion of the dendrimer band. Insufficient washing may cause the gel to show some dark precipitates when stained with coomassie blue stain (shown in the Supporting Information, p S11). Students use their cell phones C
DOI: 10.1021/acs.jchemed.8b00184 J. Chem. Educ. XXXX, XXX, XXX−XXX
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or tablets to obtain a gel picture. From the image, the distance traveled by each band (measured from a reference line to the middle of the darkest portion of a band) is measured. The migration distance is then plotted against the pI of the standard protein (obtained from the vendor) to generate a calibration curve. An example of a calibration plot is shown in Figure 3.
Laboratory Experiment
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00184. Prelaboratory material, CAS numbers, worked examples on predicting the pI of ribonuclease A and dendrimer, and details on the IEF protocol for instructors (PDF, DOC)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
A. Sharma: 0000-0002-9293-2459 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank the stockroom personnel, especially Kerin Scanlon, for preparation of the solutions and hazardous waste bottles used for this and other laboratories for the Bioanalytical Chemistry course.
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Figure 3. Plot between the migration distance of the standard proteins against their isoelectric points.
REFERENCES
(1) Benson, J. E. Viscometric determination of the isoelectric point of a protein. J. Chem. Educ. 1963, 40, 468−469. (2) Heaney, A.; Weller, D. L. Isoelectric pH of hemoglobin and cytochrome c by electrofocusing. J. Chem. Educ. 1970, 47, 724−726. (3) D’Andrea, G.; DiNicolantonio, G. A graphical approach to determine the isoelectric point and charge of small peptides from pH0 to 14. J. Chem. Educ. 2002, 79, 972−975. (4) Maldonado, A. A.; Ribeiro, J. M.; Sillero, A. Isoelectric point, electric charge, and nomenclature of the acid-base residues of proteins. Biochem. Mol. Biol. Educ. 2010, 38, 230−237. (5) Sims, P. A. Use of a spreadsheet to calculate the net charge of peptides and proteins as a function of pH: an alternative to using “canned” programs to estimate the isoelectric point of these important biomolecules. J. Chem. Educ. 2010, 87, 803−808. (6) Kannan, R. M.; Nance, E.; Kannan, S.; Tomalia, D. A. Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications. J. Intern. Med. 2014, 276, 579−617. (7) Esfand, R.; Tomalia, D. A. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discovery Today 2001, 6, 427−436. (8) Copeland, R. A. Methods for Protein Analysis: A Practical Guide to Laboratory Protocols, 1st ed.; Chapman & Hall: London, U.K., 1994; pp 79−86. (9) Upadhaya, S. K.; Swanson, D. R.; Tomalia, D. A.; Sharma, A. Analysis of polyamidoamine dendrimers by isoelectric focusing. Anal. Bioanal. Chem. 2014, 406, 455−458. (10) UniProt. https://www.uniprot.org/ (accessed June 2018). (11) Protein Information Resource. https://pir.georgetown.edu/ pirwww/search/ (accessed June (accessed July 2018). (12) Baker, J. R.; Quintana, A.; Piehler, L.; Banazak-Holl, M.; Tomalia, D.; Raczka, E. The synthesis and testing of anti-cancer nanodevices. Biomed. Microdevices 2001, 3, 61−69. (13) Baudino, T. A. Targeted cancer therapy: The next generation of cancer treatment. Curr. Drug Discovery Technol. 2015, 12, 3−20.
From the plot, the linear regression equation is obtained, and the pI values of the unknown protein and dendrimer are calculated. If the unknown protein is hen egg white lysozyme, the predicted, experimentally found, and vendor pI values are 10, 11.5, and 11.4, respectively (students who understand the proper use of calibration plots should mention in their experimental writeup that the calibration plot is assumed to be linear to pI of 11.4 since the protein standard with the highest pI is only 10.5). The predicted pI for the dendrimer is 8.6 while the typical value experimentally determined from student gels is 9.0.
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SUMMARY Using medical examples that are rarely discussed in a typical biochemistry course, using mathematical formulas to predict pI, and preparing and running an actual IEF gel help students appreciate the difficult concept of isoelectric point and its practical importance. Students also better appreciate the nature of proteins by running the IEF of a nanomolecule and proteins simultaneously; we received a favorable comment from students regarding the use of databases for determining protein structures. Student understanding of the lab and background theory was evaluated from • Problem set (open-book) performed during the lab as a group (average score of 80%) • Individual quiz (closed-book) given the following week (with an average score of 90%) • Native PAGE experiment (the following week) where students run proteins and the dendrimer under acidic conditions and interpret their relative migration data with the help of pI values D
DOI: 10.1021/acs.jchemed.8b00184 J. Chem. Educ. XXXX, XXX, XXX−XXX
