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A simple laboratory experiment to demonstrate the interaction of proteins bearing glycosylphosphatidylinositol anchors with liposomes
Biochemical Education ELSEVIER
Biochemical Education 27 (1999) 41-44
A single protein research integrated advanced biochemistry laboratory course: design and general outline Bal Ram Singh D~7~anment of'Chemist O' and Biochemisto; 285 OM Westport, Univet:~'ity +~f'Massachusetts, Dartmouth. MA 02747, USA
Abstract
An advanced biochemistry laboratory has been designed to focus on a detoxifying enzyme, glutathione-S-transferase, which is involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs), pesticides, herbicides, and other electrophilic xenobiotic compounds. The enzyme is known to catalyze conjugation of glutathione to xenobiotics, which makes them water-soluble so that they can be easily discarded through further metabolism and excretion. About two-thirds of the laboratory course incorporates nine advanced biochemical techniques, all focused to analyze various chemical characteristics of the glutathione-S-transferase. The remaining third of the semester time students work on a project that involves application of all the newly acquired techniques to solve a biochemical problem that encompasses the same detoxifying enzyme. © 1999 IUBMB. Published by Elsevier Science Ltd. All rights reserved.
1. Introduction
2. The field and the course
Biochemistry laboratory courses at undergraduate Icvel are essential to the learning and practicing of chemical analysis and the understanding of biological processes. Typical biochemistry courses utilize 'cookbook' experiments to introduce handling and analysis of biological samples using chemical techniques. In recent years, variations have been introduced whereby more than one set of experiments have been integrated to teach interconnectedness of techniques [1,2]. However, a real research-based biochemistry laboratory course has not been dcveloped to this author's knowledge. In our Department, a research-based biochemistry laboratory course has been practised for several years, except that in the past the experiments were not focused on a molecule. In the expereicnce of the author, this focus helps students learn biochemical techniques in context, and it allows a better integration with research projects. A general description of design and outline of the course is presented in this article, with a plan to communicate detailed experimental set-up and procedures in the future on a group of related experiments at a time.
Biochemistry encompasses a vast number of chemical processes to yield the basis of a biological phenomenon. Students generally need a high level of concentration and critical thinking to relate several interconnected topics. While biological processes easily attract students' attention and enthusiasm for the sake of both curiosity to learn about ourselves (living beings) and a likely opportunity to improve the quality of life, the delicate nature of biological samples generally requires a greater hands-on experience for students to learn and perform chemical analysis of such samples. Generally, all chemical techniques are applicable to biological samples but they usually require substantial modifications and adaptations to be applied to understand significant biological processes. Therefore, an advanced biochemistry laboratory course is necessary to introduce chemical techniques that are commonly used to analyze biological samples. Biochemistry laboratory courses tend to be limited in their depth and are scattered in terms of their content, primarily because of the vast number of biochemical processes involving a wide range of substances (e.g. lipids, nucleic acids, carbohydrates and proteins) which
03117-4412/98/$19.(10 + (1.(10 © 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved. PlI" ~ , [ I R l | 7 - 4 4 1 ' 2 ( Q g l t ' | [ 1 2 1 I-~
42
B.R. Singh/Biochemical Education 27 (1999) 41-44
are further complicated due to variations in organisms (e.g. bacteria, plants, and animals). Biochemistry laboratory textbooks currently available describe experiments such that a given 'cookbook' experimental system is suitable for demonstrating a particular technique. For example, in a standard Experimental Biochemistry textbook, enzyme kinetics experiment may involve egg lysozyme, but for enzyme inhibition analysis it may be lactate dehydrogenase. In the same book, the gel filtration technique may be described using immunoglobulin G as a biological sample. In such cases, while students may succeed in the 'cookbook" exercise because a given biological system is the most suitable sample to demonstrate a technique, they lose the connection between different experimental exercises. Their ability to understand the design and execution of experiments remains limited, and their potential to utilize these techniques to solve any real-world problem remains minimal. An additional problem with biochemistry laboratory courses is that the lecture class material is not always easy to directly practice in the laboratory because of the sophisticated instruments required and the substantial amount of time needed to complete experiments. One way to alleviate this is to teach a biochemistry laboratory course that is on a research topic-based system and could involve several commonly used biochemical techniques. Currently, there is no textbook available to carry out such an exercise. Therefore, the approach must be tested on an experimental basis, and if successful, it must be propagated through literature dissemination.
3. A model and initiative
We have developed a research-based Biochemistry Laboratory course that engages students in the learning of laboratory skills focused on solution of a real science problem. A research problem is generally chosen which is related to a significant biological question that can easily be understood by students with one semester of biochemistry knowledge. The project that we have tried relates to the analysis of glutathione-S-transferase (GST) of marine organisms from a local harbor which has been contaminated with polychlorinated biphenyls. New Bedford harbor (6 miles from the UMass Dartmouth campus) is a super-fund site for PCB pollution. Any project related to the PCB pollution draws absolutc attention from students because of the publicity the super-fund site has received in the local media. The enzyme (GST) has several desirable features to be used for an undergraduate project in a biochemical laboratory set-up. It is a relatively small protein (25 kDa) that exists as a dimer in solution. It is an enzyme whose genetic expression is induced by organic pollutants including PCBs. It specifically binds to glutathione so that affinity chromatography may be used for its purifica-
tion. The enzyme cxists in several isomeric forms which differ in isoelectric points that can bc exploited for ion-exchange chromatography. Finally, it is inhibited by several polyaromatic hydrocarbons (PAH), thus allowing varying kinetic analysis. We exploit biochemically relevant features of this enzyme to teach several biochemical techniques and to provide students with independent research projects for evaluating glutathione-S-transfcrascs from different marinc animals such as clams, oysters, quahogs, scallops, and other organisms. The availability of different organisms makcs new projects available evcry year. Our hmg term goal is to collaborate with the biology faculty to coordinate one of their laboratories with a Biochemistry Laboratory course so that we can include nucleic acid chemistry experiments on glutathione-S-transferascs from local marine animals to understand the role of marine pollution in the genetic expression of this enzyme as well. Currently, this approach in its present form seems to have created enthusiasm in most of the students taking this course. The approach can be applied in any part of the world because GST is ubiquitous in all organisms ranging from prokaryotes to mammals, and its gene expression is responsive to environmental conditions including pollution [3-6]. The approach has cited tremendous enthusiasm in students because not only do they learn laboratory techniques, but they also get a chance to practice them to solve a real local environmental problem.
4. Design
The course is designed to focus on the GST which is a detoxifying enzyme involved in the metabolism of PAHs, pesticides, herbicides, and other electrophilic xenobiotic compounds. The enzyme is known to catalyze the conjugation of glutathione to xenobiotics, which makes them water soluble so that they can be easily disposed of through further metabolism and excretion (Fig. 1). About two-thirds of the laboratory course incorporates nine advanced biochemical techniques, all
C1
SG
ff~ ~ A l NO2
NO2
+ GSH
+ GST - - ~ m D "~
NO2 + HCI
Glutathione
NO2 CDNB CDNB-Glutathioneconjugate Fig. 1. Schematicsof the chemicalreactionbetweenCDNBand glutathione catalyzedby glutathione-S-translerase.
B.R. Singh/Biochernical Education 27 (1999) 41-44
focused to analyze various chemical characteristics of the glutathione-S-transferase (GST). The remaining third of the semester time, students work on a project that involves application of all the newly acquired techniques to solve a biochemical problem that encompasses the same detoxifying enzyme. Last year, students decided to analyze the enzyme from a local shellfish species, quahog, obtained from a superfund site for PCB (polychlorinated biphenyls) pollution. Because of the link of the course to the local superfund environmental site-related problems, students find the whole course exciting and relevant. The project was developed keeping several points in mind. Glutathione-S-transferases are well known to be important for biomedical reasons because of their relationship to cancer in animals and humans, and for environmental reasons, as GSTs are responsible for a large part of xenobiotic metabolism. The enzyme being a protein allows application of all protein techniques to study its characteristics. In the literature, GSTs are classified based on their isoelectric points, thus providing students with a more interesting reason to determine the enzyme's isoelectric point (pl) so that they can classify it. Of course, pl is also used to plan ion-exchange chromatography. The enzyme exists as an oligomer which allows students to observe differences in the molecular size determined with the use of sodium dodecyl sulfate polyacrylamide gel electorphoresis and size-exclusion chromatography. GSTs are two-substrate enzymes, and are inhibited by a variety of biological metabolites such as bilirubin, heroin, anthocyanin, etc. providing ample opportunity to explore various enzyme kinetic issues.
5. Course outline
5.1. CHM 414: Biochemical techniques Lecture: M 1:00-1:50 pm; Laboratory: M 2:00-4:50 pm and T 1:00-3:50 pm, Text: Experimental Biochemis'try by J. Stenesh (1984), Allyn and Bacon, Boston, was used as a text to refer to fundamental topics. However, handouts with more specific instructions were provided where needed. In addition, Biochemical Calculations, by I.H. Segal, John Wiley and Sons, New York, was recommended to be useful in the course. Extensive supplementary course material was provided to include hand-outs, literature references and reprints. Material requirements: Laboratory Notebook with duplicate pages and goggles. Lab coats were also recommended. Grading: Final grades were awarded based on class quiz (5%), lab performance and lab reports (70%) and on the outcome of the final research project (25%). Formal lab reports (neatly written or typed) were
43
required for all the experiments including the research project.
5.2. Course outline and tentative schedule Wcck 1: Introduction to the laboratory and literature search and buffer preparation Objective: Library literature search to obtain biochemically relevant information on glutathioneS-transferases. Examples: Group 1--Metabolic substrates of glutathione-S-transferases; Group 2--Effect of environmental factors on glutathione-S-transferases; Group 3--Role of glutathione-S-transferases in human or animal ageing; Group 4--Plant senescence and glutathione-S-transferases. Week 2: Spectroscopic determination of protein pK Objective: Demonstrate the use of absorption spectroscopy for pKa determination. Observe variation in the pKa of Tyr side chains in a protein (glutathioneS-transferases) compared with those in free aqueous solution. Calculate the number of ionized Tyr residues in glutathione-S-transferases. Week 3: Protein estimation Objective: Use three different common methods of protein assay (Bradford, Lowry and spectroscopic) to determine absorption extinction coefficient of a protein, glutathione-S-transferases. Week 4: Discussion and catch-up Week 5: Polyacrylamide gel electrophoresis Objective: Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate molecular weight of a protein, glutathione-S-transferases. Week 6: Isoelectric focusing of protein(s) Objective: use isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) to estimate pl of a protein, and classify the given glutathioneS-transferases. Weck 7: Size exclusion chromatography Objective: Determine the native molecular weight of a protein, glutathione-S-transferases, using size exclusion column chromatography, and compare your results with the molecular weight obtained from SDS-PAGE. Week 8: Affinity chromatography Objective: Purification of glutathione-S-transferases using glutathione agarose affinity column chromatography. Obtain pure homogeneous glutathioneS-transferases (GST) from oat plants or Tetrahymena (examples). Week 9: lmmunochemical technique-ELISA
44
B.R. Singh/Biochemical Education 27 (1999) 41-44
Objective: Examine the relationship between glutathione-S-transferases from two sources with a polyclonal antibody using enzyme-linked immunosorbent assay (ELISA). Week 10: Enzyme kinetics Objectives: Estimate Km and V,..... of glutathioneS-transferases using glutathione (GSH) and 1-chloro2,4-dinitrobenzene (CDNB) as substrates. Weeks 11-15: Isolation and biochemical characterization of glutathione-S-transferases from scallops, oats, quahogs or cranberries (examples). Objectives: Utilizing all the techniques learnt in the course, isolate, purify and characterize glutathioneS-transferases for its absorption extinction coefficient, subunit structure, pl, enzyme kinetic parameters, and relationship to glutathione-S-transferases from other sources. Write your results in the form of a manuscript.
chain reaction, and recombinant gene expression has become common in workplace, as well as in graduate research. The monomeric subunit of the GST has a size of 25-30 kDa, which is small enough for genetic manipulation. Additionally, since the GST gene expression is responsive to environmental conditions, experiments related to gent expression can be designed using this system. In the second semester, students will have full opportunity to explore biochemical techniqucs on a wellcoordinated research project involving the same enzyme they would have had experience with in the first semester. This will permit learning and practising of these techniques in context. Extended time in the semester for the application of techniques to a research project will also allow me to teach formally several other techniques such as extraction of proteins, centrifugation, salting out, dialysis, protein concentration, etc., that are not currently taught in the course but are mentioned informally during the course of research project weeks.
6. Problems and solutions Acknowledgements
While students have enjoyed the 'integrated research approach' to the teaching of the biochemistry laboratory course in the past two years, they have repeatedly suggested that more time is needed to complete the research project portion of the course. I have observed myself that while the 'cookbook' set of course experiments works very well, application of the same techniques in the research project (where students have to design all the details themselves with only minimum input from the instructor) does not produce successful results at least from students' first attempts. With limited time available in the semester, students in the past have been able to succeed in applying their techniques only when they continued their experiments over part of the summer months. Therefore, I have considered and discussed with my colleagues the idea of introducing a second semester of the course which will allow me to introduce a couple of molecular biology/nucleic acid techniques in the first semester. A critical aspect of modern biochemistry, nucleic acid analysis and manipulation using molecular biology techniques, has not been covered in thc past in our biochemistry laboratory course. Part of the reason has been the lack of time in a one-semester course. Techniques such as DNA isolation, cloning, polymerase
The author would like to thank his colleagues in the Department, especially J.D. Smith and W.L. Dills, for their support and ideas on this project. Financial support from The Camille and Henry Dreyfus Foundation is greatly acknowledged.
References [1] J.G. Harman, J.A. Anderson, R.A. Nakashima, R.W. Shaw, An integrated approach to the undergraduate biochemistry laboratory. J. Chem. Educ. 72 (1995) 641-645. [2] A.J. Wolfson, M.L. Hall, T.R. Branham, An integrated biochemistry laboratory, including molecular modelling. J. Chem. Educ. 73 (1996) 1026-1(/28. [3] B. Manncrvik, U.H. Daniclson, Glutathionc-S-transferases structure and catalytic activity. CRC Critical Rev. Biochemistry 23 (1998) 283-337. [4J T. Prestera, W.D. Holtzclaw, Y. Shang, P. Talalay, Chemical and molecular regulation of enzymes that detoxify carcinogens. Proc. Natl. Acad. Sci. USA 90 (1993) 2965-2969. [5] T.H. Rushmore. C.B. Pickett, Glutathione-S-transfcrases, structure, regulation, and therapeutic implications. J. Biol. Chem. 268 (1993) 11475-11478. [6] E.A. Vandewaa, C.K. Campbell, K. O'Leary, J.W. Tracy, Induction of Schistosoma mansoni glutathione-S-transferases by xenobiotics. Arch. Biochem. Biophys. 3(13 (1993) 15-21.
Biochemical Education 27 (1999) 41-44
A single protein research integrated advanced biochemistry laboratory course: design and general outline Bal Ram Singh D~7~anment of'Chemist O' and Biochemisto; 285 OM Westport, Univet:~'ity +~f'Massachusetts, Dartmouth. MA 02747, USA
Abstract
An advanced biochemistry laboratory has been designed to focus on a detoxifying enzyme, glutathione-S-transferase, which is involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs), pesticides, herbicides, and other electrophilic xenobiotic compounds. The enzyme is known to catalyze conjugation of glutathione to xenobiotics, which makes them water-soluble so that they can be easily discarded through further metabolism and excretion. About two-thirds of the laboratory course incorporates nine advanced biochemical techniques, all focused to analyze various chemical characteristics of the glutathione-S-transferase. The remaining third of the semester time students work on a project that involves application of all the newly acquired techniques to solve a biochemical problem that encompasses the same detoxifying enzyme. © 1999 IUBMB. Published by Elsevier Science Ltd. All rights reserved.
1. Introduction
2. The field and the course
Biochemistry laboratory courses at undergraduate Icvel are essential to the learning and practicing of chemical analysis and the understanding of biological processes. Typical biochemistry courses utilize 'cookbook' experiments to introduce handling and analysis of biological samples using chemical techniques. In recent years, variations have been introduced whereby more than one set of experiments have been integrated to teach interconnectedness of techniques [1,2]. However, a real research-based biochemistry laboratory course has not been dcveloped to this author's knowledge. In our Department, a research-based biochemistry laboratory course has been practised for several years, except that in the past the experiments were not focused on a molecule. In the expereicnce of the author, this focus helps students learn biochemical techniques in context, and it allows a better integration with research projects. A general description of design and outline of the course is presented in this article, with a plan to communicate detailed experimental set-up and procedures in the future on a group of related experiments at a time.
Biochemistry encompasses a vast number of chemical processes to yield the basis of a biological phenomenon. Students generally need a high level of concentration and critical thinking to relate several interconnected topics. While biological processes easily attract students' attention and enthusiasm for the sake of both curiosity to learn about ourselves (living beings) and a likely opportunity to improve the quality of life, the delicate nature of biological samples generally requires a greater hands-on experience for students to learn and perform chemical analysis of such samples. Generally, all chemical techniques are applicable to biological samples but they usually require substantial modifications and adaptations to be applied to understand significant biological processes. Therefore, an advanced biochemistry laboratory course is necessary to introduce chemical techniques that are commonly used to analyze biological samples. Biochemistry laboratory courses tend to be limited in their depth and are scattered in terms of their content, primarily because of the vast number of biochemical processes involving a wide range of substances (e.g. lipids, nucleic acids, carbohydrates and proteins) which
03117-4412/98/$19.(10 + (1.(10 © 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved. PlI" ~ , [ I R l | 7 - 4 4 1 ' 2 ( Q g l t ' | [ 1 2 1 I-~
42
B.R. Singh/Biochemical Education 27 (1999) 41-44
are further complicated due to variations in organisms (e.g. bacteria, plants, and animals). Biochemistry laboratory textbooks currently available describe experiments such that a given 'cookbook' experimental system is suitable for demonstrating a particular technique. For example, in a standard Experimental Biochemistry textbook, enzyme kinetics experiment may involve egg lysozyme, but for enzyme inhibition analysis it may be lactate dehydrogenase. In the same book, the gel filtration technique may be described using immunoglobulin G as a biological sample. In such cases, while students may succeed in the 'cookbook" exercise because a given biological system is the most suitable sample to demonstrate a technique, they lose the connection between different experimental exercises. Their ability to understand the design and execution of experiments remains limited, and their potential to utilize these techniques to solve any real-world problem remains minimal. An additional problem with biochemistry laboratory courses is that the lecture class material is not always easy to directly practice in the laboratory because of the sophisticated instruments required and the substantial amount of time needed to complete experiments. One way to alleviate this is to teach a biochemistry laboratory course that is on a research topic-based system and could involve several commonly used biochemical techniques. Currently, there is no textbook available to carry out such an exercise. Therefore, the approach must be tested on an experimental basis, and if successful, it must be propagated through literature dissemination.
3. A model and initiative
We have developed a research-based Biochemistry Laboratory course that engages students in the learning of laboratory skills focused on solution of a real science problem. A research problem is generally chosen which is related to a significant biological question that can easily be understood by students with one semester of biochemistry knowledge. The project that we have tried relates to the analysis of glutathione-S-transferase (GST) of marine organisms from a local harbor which has been contaminated with polychlorinated biphenyls. New Bedford harbor (6 miles from the UMass Dartmouth campus) is a super-fund site for PCB pollution. Any project related to the PCB pollution draws absolutc attention from students because of the publicity the super-fund site has received in the local media. The enzyme (GST) has several desirable features to be used for an undergraduate project in a biochemical laboratory set-up. It is a relatively small protein (25 kDa) that exists as a dimer in solution. It is an enzyme whose genetic expression is induced by organic pollutants including PCBs. It specifically binds to glutathione so that affinity chromatography may be used for its purifica-
tion. The enzyme cxists in several isomeric forms which differ in isoelectric points that can bc exploited for ion-exchange chromatography. Finally, it is inhibited by several polyaromatic hydrocarbons (PAH), thus allowing varying kinetic analysis. We exploit biochemically relevant features of this enzyme to teach several biochemical techniques and to provide students with independent research projects for evaluating glutathione-S-transfcrascs from different marinc animals such as clams, oysters, quahogs, scallops, and other organisms. The availability of different organisms makcs new projects available evcry year. Our hmg term goal is to collaborate with the biology faculty to coordinate one of their laboratories with a Biochemistry Laboratory course so that we can include nucleic acid chemistry experiments on glutathione-S-transferascs from local marine animals to understand the role of marine pollution in the genetic expression of this enzyme as well. Currently, this approach in its present form seems to have created enthusiasm in most of the students taking this course. The approach can be applied in any part of the world because GST is ubiquitous in all organisms ranging from prokaryotes to mammals, and its gene expression is responsive to environmental conditions including pollution [3-6]. The approach has cited tremendous enthusiasm in students because not only do they learn laboratory techniques, but they also get a chance to practice them to solve a real local environmental problem.
4. Design
The course is designed to focus on the GST which is a detoxifying enzyme involved in the metabolism of PAHs, pesticides, herbicides, and other electrophilic xenobiotic compounds. The enzyme is known to catalyze the conjugation of glutathione to xenobiotics, which makes them water soluble so that they can be easily disposed of through further metabolism and excretion (Fig. 1). About two-thirds of the laboratory course incorporates nine advanced biochemical techniques, all
C1
SG
ff~ ~ A l NO2
NO2
+ GSH
+ GST - - ~ m D "~
NO2 + HCI
Glutathione
NO2 CDNB CDNB-Glutathioneconjugate Fig. 1. Schematicsof the chemicalreactionbetweenCDNBand glutathione catalyzedby glutathione-S-translerase.
B.R. Singh/Biochernical Education 27 (1999) 41-44
focused to analyze various chemical characteristics of the glutathione-S-transferase (GST). The remaining third of the semester time, students work on a project that involves application of all the newly acquired techniques to solve a biochemical problem that encompasses the same detoxifying enzyme. Last year, students decided to analyze the enzyme from a local shellfish species, quahog, obtained from a superfund site for PCB (polychlorinated biphenyls) pollution. Because of the link of the course to the local superfund environmental site-related problems, students find the whole course exciting and relevant. The project was developed keeping several points in mind. Glutathione-S-transferases are well known to be important for biomedical reasons because of their relationship to cancer in animals and humans, and for environmental reasons, as GSTs are responsible for a large part of xenobiotic metabolism. The enzyme being a protein allows application of all protein techniques to study its characteristics. In the literature, GSTs are classified based on their isoelectric points, thus providing students with a more interesting reason to determine the enzyme's isoelectric point (pl) so that they can classify it. Of course, pl is also used to plan ion-exchange chromatography. The enzyme exists as an oligomer which allows students to observe differences in the molecular size determined with the use of sodium dodecyl sulfate polyacrylamide gel electorphoresis and size-exclusion chromatography. GSTs are two-substrate enzymes, and are inhibited by a variety of biological metabolites such as bilirubin, heroin, anthocyanin, etc. providing ample opportunity to explore various enzyme kinetic issues.
5. Course outline
5.1. CHM 414: Biochemical techniques Lecture: M 1:00-1:50 pm; Laboratory: M 2:00-4:50 pm and T 1:00-3:50 pm, Text: Experimental Biochemis'try by J. Stenesh (1984), Allyn and Bacon, Boston, was used as a text to refer to fundamental topics. However, handouts with more specific instructions were provided where needed. In addition, Biochemical Calculations, by I.H. Segal, John Wiley and Sons, New York, was recommended to be useful in the course. Extensive supplementary course material was provided to include hand-outs, literature references and reprints. Material requirements: Laboratory Notebook with duplicate pages and goggles. Lab coats were also recommended. Grading: Final grades were awarded based on class quiz (5%), lab performance and lab reports (70%) and on the outcome of the final research project (25%). Formal lab reports (neatly written or typed) were
43
required for all the experiments including the research project.
5.2. Course outline and tentative schedule Wcck 1: Introduction to the laboratory and literature search and buffer preparation Objective: Library literature search to obtain biochemically relevant information on glutathioneS-transferases. Examples: Group 1--Metabolic substrates of glutathione-S-transferases; Group 2--Effect of environmental factors on glutathione-S-transferases; Group 3--Role of glutathione-S-transferases in human or animal ageing; Group 4--Plant senescence and glutathione-S-transferases. Week 2: Spectroscopic determination of protein pK Objective: Demonstrate the use of absorption spectroscopy for pKa determination. Observe variation in the pKa of Tyr side chains in a protein (glutathioneS-transferases) compared with those in free aqueous solution. Calculate the number of ionized Tyr residues in glutathione-S-transferases. Week 3: Protein estimation Objective: Use three different common methods of protein assay (Bradford, Lowry and spectroscopic) to determine absorption extinction coefficient of a protein, glutathione-S-transferases. Week 4: Discussion and catch-up Week 5: Polyacrylamide gel electrophoresis Objective: Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate molecular weight of a protein, glutathione-S-transferases. Week 6: Isoelectric focusing of protein(s) Objective: use isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) to estimate pl of a protein, and classify the given glutathioneS-transferases. Weck 7: Size exclusion chromatography Objective: Determine the native molecular weight of a protein, glutathione-S-transferases, using size exclusion column chromatography, and compare your results with the molecular weight obtained from SDS-PAGE. Week 8: Affinity chromatography Objective: Purification of glutathione-S-transferases using glutathione agarose affinity column chromatography. Obtain pure homogeneous glutathioneS-transferases (GST) from oat plants or Tetrahymena (examples). Week 9: lmmunochemical technique-ELISA
44
B.R. Singh/Biochemical Education 27 (1999) 41-44
Objective: Examine the relationship between glutathione-S-transferases from two sources with a polyclonal antibody using enzyme-linked immunosorbent assay (ELISA). Week 10: Enzyme kinetics Objectives: Estimate Km and V,..... of glutathioneS-transferases using glutathione (GSH) and 1-chloro2,4-dinitrobenzene (CDNB) as substrates. Weeks 11-15: Isolation and biochemical characterization of glutathione-S-transferases from scallops, oats, quahogs or cranberries (examples). Objectives: Utilizing all the techniques learnt in the course, isolate, purify and characterize glutathioneS-transferases for its absorption extinction coefficient, subunit structure, pl, enzyme kinetic parameters, and relationship to glutathione-S-transferases from other sources. Write your results in the form of a manuscript.
chain reaction, and recombinant gene expression has become common in workplace, as well as in graduate research. The monomeric subunit of the GST has a size of 25-30 kDa, which is small enough for genetic manipulation. Additionally, since the GST gene expression is responsive to environmental conditions, experiments related to gent expression can be designed using this system. In the second semester, students will have full opportunity to explore biochemical techniqucs on a wellcoordinated research project involving the same enzyme they would have had experience with in the first semester. This will permit learning and practising of these techniques in context. Extended time in the semester for the application of techniques to a research project will also allow me to teach formally several other techniques such as extraction of proteins, centrifugation, salting out, dialysis, protein concentration, etc., that are not currently taught in the course but are mentioned informally during the course of research project weeks.
6. Problems and solutions Acknowledgements
While students have enjoyed the 'integrated research approach' to the teaching of the biochemistry laboratory course in the past two years, they have repeatedly suggested that more time is needed to complete the research project portion of the course. I have observed myself that while the 'cookbook' set of course experiments works very well, application of the same techniques in the research project (where students have to design all the details themselves with only minimum input from the instructor) does not produce successful results at least from students' first attempts. With limited time available in the semester, students in the past have been able to succeed in applying their techniques only when they continued their experiments over part of the summer months. Therefore, I have considered and discussed with my colleagues the idea of introducing a second semester of the course which will allow me to introduce a couple of molecular biology/nucleic acid techniques in the first semester. A critical aspect of modern biochemistry, nucleic acid analysis and manipulation using molecular biology techniques, has not been covered in thc past in our biochemistry laboratory course. Part of the reason has been the lack of time in a one-semester course. Techniques such as DNA isolation, cloning, polymerase
The author would like to thank his colleagues in the Department, especially J.D. Smith and W.L. Dills, for their support and ideas on this project. Financial support from The Camille and Henry Dreyfus Foundation is greatly acknowledged.
References [1] J.G. Harman, J.A. Anderson, R.A. Nakashima, R.W. Shaw, An integrated approach to the undergraduate biochemistry laboratory. J. Chem. Educ. 72 (1995) 641-645. [2] A.J. Wolfson, M.L. Hall, T.R. Branham, An integrated biochemistry laboratory, including molecular modelling. J. Chem. Educ. 73 (1996) 1026-1(/28. [3] B. Manncrvik, U.H. Daniclson, Glutathionc-S-transferases structure and catalytic activity. CRC Critical Rev. Biochemistry 23 (1998) 283-337. [4J T. Prestera, W.D. Holtzclaw, Y. Shang, P. Talalay, Chemical and molecular regulation of enzymes that detoxify carcinogens. Proc. Natl. Acad. Sci. USA 90 (1993) 2965-2969. [5] T.H. Rushmore. C.B. Pickett, Glutathione-S-transfcrases, structure, regulation, and therapeutic implications. J. Biol. Chem. 268 (1993) 11475-11478. [6] E.A. Vandewaa, C.K. Campbell, K. O'Leary, J.W. Tracy, Induction of Schistosoma mansoni glutathione-S-transferases by xenobiotics. Arch. Biochem. Biophys. 3(13 (1993) 15-21.