- Email: [email protected]
Introduction to Research: An Undergraduate Biochemistry Laboratory
18 approached regarding the availability of the bank by several undergraduate departments which teach large biochemistry classes. Currently the contract which subscribers sign precludes their distribution of all or part of the bank beyond their department. This has been interpreted to mean that those departments which teach undergraduates may use the bank for their students but that other undergraduate departments which are not affiliated with a medical department of biochemistry may not have access to the bank. Because of a perceived need on the part of some undergraduate departments for a large pool of excellent test questions, the Editorial Board, in conjunction with AMSDB, will re-evaluate during 1981 the distribution policy to determine whether all or a part of the bank could or should be made available for wider distribution. The potential advantage to the new subscribers would be the access to a large pool of test items. In return, the wider distribution would enable the Editorial Board to undertake some of the educational and evaluative projects which have been described earlier. A potential drawback which must be fully explored is the nature of the impact on undergraduate biochemical training that this clinically-oriented question bank would have. Thus, there would seem to be a sufficient number of advantages to the parties concerned to merit further cautious exploration in this area. The Editor-inChief would like to receive in writing an indication of the interest of undergraduate departments in the use of this type of question bank. Based on the degree of interest and the perception of the advantages and disadvantages to the biochemical community at large, the decision will be reached by the membership of AMSDB on the wider distribution of this question bank. Note The addresses of the members of the Editorial Board are as follows: JAP, Department of Biochemistry, The University of Texas Health Science Center at Dallas, 5323 Hines Boulevard, Dallas, TX 75235; CCA, Department of Biochemistry, University of Tennessee Center for the Health Sciences, Memphis, TN 38163; JB, Department of Biochemistry, Hahnemann Medical College, 235 North 15th Street, Philadelphia, PA 19102; JPB, Department of Biochemistry, University of Oregon Health Science Center, 3181 SW Sam Jackson Park, Portland, OR 97201; JLF, Department of Biochemistry, Michigan State University, East Lansing, MI 48824; ABR, Department of Biochemistry, The University of Kansas Medical Center, Rainbow Boulevard at 39th, Kansas City, KS 66103; and LWS, Department of Biochemistry, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29403. Please address all correspondence to: Dr Julian A Peterson, Editor-in-Chief, MBQB, Department of Biochemistry, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235.
Announcement Eighth International Conference on 'Improving University Teaching', July 14-17, 1982, in West Berlin The deadline for the receipt of papers is 1st February 1982, and for registration 1st April 1982, otherwise a late registration fee is charged. Further details may be obtained from: 'Improving University Teaching' University of Maryland University College University Boulevard at Adelphi Road College Park, Maryland 20742, USA Papers should be sent to this address, but registrations may be sent to the above address or to 'Improving University Teaching', University of Maryland, Im Bosseldorn 30, 6900 Heidelberg, Federal Republic of Germany. BIOCHEMICAL EDUCATION
10(1)
1982
Introduction to Research: An Undergraduate Biochemistry Laboratory CLARENCE H SUELTER
Department of Biochemistry Michigan State Universi~. East Lansing, Michigan, USA Introduction The successful description of the chemistry of biological tissues requires the development and use of unique and sophisticated techniques and instrumentation. Thus one might argue that biochemistry undergraduate laboratory courses should continually be modified to provide experience in the various new technologies. After reflection though, most of us realize that there is not sufficient time to offer undergraduate students detailed experience in all techniques being used in biochemical research laboratories. Laidler 1 stated it succinctly: 'there is too much to know'. Is there a solution to this problem? What should be done? What can be done?
Structured formats It is my thesis that undergraduate laboratory courses with structured formats in which students follow printed protocols using prepared reagents to complete an experiment do not completely meet the basic needs of students entering research laboratories. On the one hand I believe that this format is useful for the introduction of basic techniques to students. By using prepared reagents, students will not be simultaneously challenged by both the techniques used in an experiment and the need to design and prepare the reagents for the experiments. In my experience, this format has proved successful and continues to be used in the teaching of many experiences that science students need. The danger of a structured format, on the other hand, is the tendency to expose students to sophisticated instruments and methodologies at the expense of learning the experimental design. This tendency is reinforced by the desire of students to seek this exposure. Many students finish this course without the background needed to design a simple experiment. They lack the sense of where to start. What volume of reagents should be prepared? What concentration? Should temperature and ionic strength be controlled? What about the pH of a reaction? What is an adequate control? This lack of experience in identifying the basic elements of an experimental design often results in the failure of many students to initiate a successful research career. As pointed out by Laidler I 'competence in science depends less on how much we know than what we can do with the scientific problems with which we are faced'.
Research exercises This paper describes an undergraduate laboratory with exercises designed to introduce students to the research process. Each student works individually and with assistance develops an experimental design, decides on the amount and concentration of each reagent needed and prepares the reagents. After completion of each exercise, the student submits a report modeled after a scientific paper. This is carefully read and constructive criticism is provided.
19 In my view, the topic of the laboratory exercises is of little concern. Our experience involves a course centred in enzymology as outlined on Scheme 1. No doubt there are other scenarios that could be designed. It is our belief, however, that all students of biochemistry, regardless of their primary interest within the discipline, will profit from a working knowledge of the structure and function of enzymes. The single topic approach also allows one to build a series of experiments proceeding from a less complex to a more complex experimental design. The course assumes exposure to one year of didactic biochemistry (90 lectures) and previous basic laboratory experience. Experience in physical chemistry is not essential but is helpful. The first six periods of instruction after the introductory lecture provide students with some basic fundamentals of research. These include data collection and analysis including the importance of statistics, preparation of buffers, the use of microtechniques and their importance, keeping a good research notebook, designing a useful protocol, the problems
involved in assaying an enzyme and the elements of writing a good scientific paper. The fundamentals of ultraviolet-visible spectrophotometry and pH are also reviewed. After the first six periods, students begin a study of yeast alcohol dehydrogenase. Each experiment is preceded by a 15-20 minute lecture available on an audiovisual cassette. Only a minimum of didactic material is reviewed. Experiments involving the determination of first- and second-order rate constants, enzyme kinetic parameters, and recombinant DNA enzymology are simulated on a microcomputer) Audiovisual lectures, the simulated experiments on a microcomputer and tutorial assistance are provided to help the student in designing an experiment, writing the protocol and completing the experiment. The protocol must be approved by an instructor before the student has permission to initiate the laboratory work. Each student is allotted a generous but fLxed time-period in which to complete the experiment, analyze the data and write a scientific laboratory report. Laboratory reports should be expected at fixed time-periods throughout the term.
Scheme 1 One of several possible sequences of topics for a laboratory course
Introduction to Biochemical Research
I
II
III
Introduction and Objectives Basic (1) (2) (3) (4) (5) (6) (7) (8) (9)
Techniques Statistical considerations in data collection and analysis Buffers in a biochemical research laboratory Microtechniques in a biochemical research laboratory Laboratory notebooks - keeping research records Protocols - What are they? Spectrophotometry Enzyme assay - Assay of yeast alcohol dehydrogenase Handling animals - Bleeding a rabbit Report - Composition and structure
Experiments (1) Structure of yeast alcohol dehydrogenase a Number of buried and exposed sulphydryl groups by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) b Determine pseudo 1st order and 2nd order rate constant for reaction (2) Function of yeast alcohol dehydrogenase a Enzymatic assay of metabolite, NAD ÷ b Equilibrium constant of yeast alcohol dehydrogenase - effect of pH (optional) c Kinetic properties of yeast alcohol dehydrogenase; K m and NAD ÷ and Ki for NADH at infinite concentration of ethanol (3) Purification, molecular weight and antigenic properties of yeast alcohol dehydrogenase a Fractional precipitation of yeast alcohol dehydrogenase from a crude yeast extract by PEG 4000 b DEAE-cellulose chromatography c Comparative elution of yeast alcohol dehydrogenase with standard proteins from a gel permeation column using high pressure liquid chromatographic techniques d Antigenic properties of yeast alcohol dehydrogenase: (i) Ouchterlony double diffusion, and (ii) determination of antibody titre (4) Recombinant DNA enzymology a Restriction of pBR 322 and XDNA with Eco RI b Ligation of restricted pBR 322 and XDNA with T4 DNA ligase c Agarose gel electrophoresis
BIOCHEMICAL EDUCATION
10(1) 1982
No of 3 h periods recommended for completion
20 A laboratory manual is provided that outlines the objectives of each experiment. Broad guidelines are provided so that the experimental protocol can be written without prior experimentation. It is necessary to hold a discussion once a week to provide a forum for group interaction in the analysis and interpretation of data.
Description of experiments Experiment I The first experiment provides students with experience in classical chemical kinetics. The number of exposed sulphydryl groups is obtained from the change in absorption at 412 nm (e = 14,100M -1 cm-1) a after reaction of excess 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) with the sulphydryl groups. Sodium dodecyl sulphate is added to unfold the protein and expose the buried residues. Generally, all sulphydryls of yeast ADH react as exposed residues in Tris/HCl buffer, pH 8 to 9. DTNB is dissolved in water containing an equivalent amount of Na2CO3. Commercial lyophilized yeast ADH is provided to each student for this and experiment 2. Experiment 2 The kinetic parameters, Km for NAD and K i for NADH, are measured by standard spectrophotometric techniques. Data are analyzed by procedures as suggested by Wilkinson4 with the assistance of a PET microcomputer. 2 Students are given literature values for Km and Ki s as an aid in designing the experiment. Experiment3 The third experiment is more complex. It requires the application of several techniques to the partial purification of yeast ADH from a crude yeast extract. Each student is provided with 30-50 ml of a crude yeast extract prepared according to a published procedure. 6 Solid cake yeast (500 g) is crumbled into liquid N2 in a Dewar. After the yeast is frozen it and the liquid N2 are poured into a Waring Blender and blended for 4 minutes in 1 minute intervals. (The blade assembly of the blender must be dry to prevent freeze-up.) One litre of 20 mM sodium phosphate, pH 7.8, is added to the fine powder and the suspension homogenized after it has thawed. This homogenate is stirred for an additional hour and centrifuged. One-fifth volume of a 2% solution of protamine sulfate is added to the supernatant which is stirred for 20 minutes at room temperature. After centrifuging and adjusting the supernatant to pH 7 with 1:1 NH4OH, twenty-millilitre aliquots are stored in plastic scintillation vials at -20 ° for distribution to the student. The first step of the purification requires the student to determine the amount of PEG 4000 (50% w/v) required to complete a fractional precipitation. PEG 4000 concentrations ranging from 0 to 20% are appropriate. After subjecting the remaining crude yeast extract to the PEG 4000 fractionation, the precipitate is dissolved in a minimum volume of 5 mM phosphate buffer, pH 7.0. Between 10 and 20 mg of protein, estimated from the absorbance at 280 nm (assume e =0.8 ml mg -~ cm-1), at 5-10 mg/ml is applied to 8-10 ml of DEAE cellulose packed in a 10 ml disposable syringe column. This is eluted with a linear gradient of sodium phosphate composed of 25 ml of 5 mM and 25 ml of 0.15 M sodium phosphate, pH 7.5. Twenty-five 2 ml fractions are collected by hand. Protein concentration is estimated from the absorbances at 280 and 260 nm. The conductance of 0.1 ml aliquots diluted to 3 ml is measured to determine the linearity of the gradient. BIOCHEMICAL EDUCATION
1 0 ( 1 ) 1982
The peak fractions containing enzyme activity can be pooled and precipitated by dialysis against saturated ammonium sulphate. The molecular weight of commercial yeast ADH is determined by comparative elution with standard proteins on a gel permeation colum using high pressure liquid chromatographic techniques. 1 Normally 6 to 8 standard proteins are used. Experimental data for the HPLC experiment can be obtained in a 2 hour period. Data for molecular weight determination may be obtained with a calibrated gel-permeation column developed under gravity flow. Depending on the design of the experiment, this approach may be more time-consuming. Early in the term, a rabbit is bled to obtain pre-immune serum, then immunized twice, once with alcohol dehydrogenase suspended in Freund's complete adjuvant followed by a second injection 3 weeks later.* The immune serum is is collected one week after the second injection. Three students use one rabbit. Each student has an opportunity to work with the animal and perform the necessary operations. Blood is collected in Petri dishes (15 cm) and allowed to clot overnight before the serum is removed. A 'V' cut in the clot with a razor-blade increases the yield of serum. The pre-and post-immune serum is subjected to an Ouchterlony double diffusion with both the commercial yeast enzyme and the partially purified enzyme. The antibody titre is obtained from a plot of enzyme activity remaining after titration of a fixed amount of antibody with enzyme.
Experiment 4 This experiment is technically more difficult. It is completed during the last four periods after students have gained experience with microtechniques. Basically each student sets up a 30/21 reaction involving digestion separately of XDNA and pBR322 plasmid vector with a restriction endonuclease, and ligation of the XDNA fragments with the restricted vector. Electrophoresis on agarose gel and staining with ethidium bromide tests the results of the ligation and gives the students experience in interpreting gel-electrophoretic data.**
Discussion Some of the major goals of this laboratory are (1) to give students an opportunity to design their experiments, ( 2 ) t o give students experience in writing their own protocols, (3) to impress upon students the importance of using microtechniques to conserve material, (4) to provide experience in using microtechniques, (5) to give students assistance and experience in interpreting data, (6) to provide experience and constructive criticism in writing scientific research papers, ( 7 ) t o provide experience and constructive criticism in maintaining a research notebook, and (8) to impress upon the students the need to be concerned with the errors involved in an experiment. The first class period of 3 hours is devoted to introducing students to the structure of the course and the requirements for its satisfactory completion. Next we compare the format of this course to a typical research laboratory and point out that experiments in an academic research laboratory are generally designed by a student in conjunction with his/her peers and~or mentor. After the experiments are completed, *Editorial Note: Readers should be cognizant of restrictions in their respective country of doing experiments with live animals. **As soon as the material becomes available, this experiment will be done with the yeast DNA for alcohol dehydrogenase.
21 the results are shared and analyzed again in conjunction with his/her peers and/or mentor. As a result we encourage students to discuss their protocols and experimental results among themselves. The weekly group discussions provide a useful forum for discussing the data. Finally it is important to note that an experiment has no meaning unless its results are communicated to the scientific community at large. All other lecture material is provided on audiovisual cassettes. Thus it is not necessary for all students to begin each experiment at the same time. In fact, to minimize the need for multiple units of major equipment, students are encouraged to stagger the start of various experiments. Each audiovisual cassette is designed to introduce the basic elements of a technique or procedure. For instance, the cassette entitled 'The Assay of an Enzyme' covers the following topics: end point and continuous assays, direct and coupled assays, assay temperature and pH, reaction equilibrium, substrate concentration, starting the reaction, measurement of initial velocity, and specific activity. The primary disadvantage of this approach involves the need for time. First students, at least during the early part of the term, require assistance in using instruments and writing protocols. Since the structure is flexible, we have provided assistance for 4-4½ hours instead of the normal 3-hour laboratory period. As the term progresses students require less and less assistance but many need the time to complete the experiments. Students also sense a time pressure to f'mish an experiment. This often results in the presentation of a minimum number of data, sometimes without replication. It is also important to realize that everyone, including students, has a tendency to procrastinate and therefore it is important to have deadlines for reports during the tenn. Students who take this course are well satisfied. They enjoy the challenge of writing their own protocols, preparing their own reagents, working individually, and the flexibility of working at their own pace. The lectures on audiovisual cassettes and the microcomputer simulations are convenient since the background material for an experiment can be reviewed and simulated immediately prior to preparing a protocol. The main complaint revolved around the need for more time to assimilate the results of an experiment. This complaint is partially rectified by the weekly discussion period in which experimental data are discussed and analyzed. The cost of this offering is not excessive. It can be minimized by emphasizing microtechniques, using plastic cuvettes, and careful monitoring of the amount of material planned for use as indicated by the protocols. The cost can be reduced by preparing ADH from yeast. Experiment 4 may also be deleted to reduce costs without seriously jeopardizing the objectives of the laboratory experiences. Good thermostarted ultraviolet-visible recording spectrophotometers are necessary for the course indicated by Scheme 1. References 1Laidler, K J (1974) JChem Education 51, 696-700 2 Suelter, C H and Hill, D (1981) J Chem Education, in the press 3 Riddles, P W, Blakeley, R L and Zerner, B (1979) Analyt Biochem 94, 75 -81 *Wilkinson, G N (1961) Biochem J 8 0 , 324-332 SSund, H and Theorell, H (1963) in 'The Enzymes', 2nd Edition, P D Boyer, H Lardy and K Myrback, editors, Academic Press, New York, pp 25-83 6 Dethmers, J K, Ferguson-Miller, S and Margoliash, E (1979) J Biol Chem 254, 11973-11981 7 Regnier, F E and Gooding, K M (1980) Analyt Blochem 103, 1-25.
BIOCHEMICAL EDUCATION
10(1) 1982
Science Research in the Universities S L BONTING
Department of Biochemistry University of Ni]megen Ni]megen, Netherlands Introduction
In order to say anything of significance on such a broad topic it will be necessary to limit severely the points to be considered. Thus, although it would be very desirable to consider science research in the Communist countries and the Third World countries, I shall limit myself to the Western world and more particularly Western Europe. Having thus limited the topic territorially, there immediately suggest themselves for consideration the tremendous growth in science research during the sixties and early seventies and the consequences of this growth: promises full'died and unf'ilfulled, effects good and bad, societal and governmental reactions favourable and unfavourable, scientists as heroes and villains. On top of this we have the radically changed economic and social conditions in the Western world since the early seventies, which are having their repercussion on science research in the universities. These aspects will be discussed under four headings: (1) organization and f'mancing; (2) science policy; (3) further developments; and (4) the responsibility of the scientists. Organization and f'mancing Information from six representative Western European countries was obtained from a recent study by Van Rossum*: Belgium, Italy, the Netherlands, Norway, United Kingdom and Switzerland. In all of these countries university research is almost completely funded by the Government, both directly (through general university funding) and indirectly (through Research Councils); private foundations play a very minor role (2% or less), in contrast to the situation in the USA. In all cases the Ministry of Education is solely or largely responsible for university funding, but in the United Kingdom the University Grants Committee budgets, receives and distributes these funds. Typical for all these countries is the large growth from the fifties to the seventies in manpower and funds for science research in general, but particularly for that in the universities. Table 1 illustrates this for the Netherlands. Responsible for this were the favourable economic conditions in Western Europe over this period, the large increase in student enrolment with subsequent increase in staff, and probably the effect of defence spending. Table 2 shows that this rapid growth was a general phenomenon in Western Europe, even though here only figures for the latter half of this period were available. When we add to this the many European joint research installations and efforts, like CERN, Euratom, ESA (European Space Agency) and EMBL (European Molecular Biology Laboratory), we can conclude that the European science research potential now equals that of the USA. Whether this is also true for scientific productivity and number of scientific 'breakthroughs' is another question! *W. van Rossum, The Organization and Financing of {ParaJ-University Research in Western Europe: A Comparative View, 1979, 132 pp, no publisher.
Announcement Eighth International Conference on 'Improving University Teaching', July 14-17, 1982, in West Berlin The deadline for the receipt of papers is 1st February 1982, and for registration 1st April 1982, otherwise a late registration fee is charged. Further details may be obtained from: 'Improving University Teaching' University of Maryland University College University Boulevard at Adelphi Road College Park, Maryland 20742, USA Papers should be sent to this address, but registrations may be sent to the above address or to 'Improving University Teaching', University of Maryland, Im Bosseldorn 30, 6900 Heidelberg, Federal Republic of Germany. BIOCHEMICAL EDUCATION
10(1)
1982
Introduction to Research: An Undergraduate Biochemistry Laboratory CLARENCE H SUELTER
Department of Biochemistry Michigan State Universi~. East Lansing, Michigan, USA Introduction The successful description of the chemistry of biological tissues requires the development and use of unique and sophisticated techniques and instrumentation. Thus one might argue that biochemistry undergraduate laboratory courses should continually be modified to provide experience in the various new technologies. After reflection though, most of us realize that there is not sufficient time to offer undergraduate students detailed experience in all techniques being used in biochemical research laboratories. Laidler 1 stated it succinctly: 'there is too much to know'. Is there a solution to this problem? What should be done? What can be done?
Structured formats It is my thesis that undergraduate laboratory courses with structured formats in which students follow printed protocols using prepared reagents to complete an experiment do not completely meet the basic needs of students entering research laboratories. On the one hand I believe that this format is useful for the introduction of basic techniques to students. By using prepared reagents, students will not be simultaneously challenged by both the techniques used in an experiment and the need to design and prepare the reagents for the experiments. In my experience, this format has proved successful and continues to be used in the teaching of many experiences that science students need. The danger of a structured format, on the other hand, is the tendency to expose students to sophisticated instruments and methodologies at the expense of learning the experimental design. This tendency is reinforced by the desire of students to seek this exposure. Many students finish this course without the background needed to design a simple experiment. They lack the sense of where to start. What volume of reagents should be prepared? What concentration? Should temperature and ionic strength be controlled? What about the pH of a reaction? What is an adequate control? This lack of experience in identifying the basic elements of an experimental design often results in the failure of many students to initiate a successful research career. As pointed out by Laidler I 'competence in science depends less on how much we know than what we can do with the scientific problems with which we are faced'.
Research exercises This paper describes an undergraduate laboratory with exercises designed to introduce students to the research process. Each student works individually and with assistance develops an experimental design, decides on the amount and concentration of each reagent needed and prepares the reagents. After completion of each exercise, the student submits a report modeled after a scientific paper. This is carefully read and constructive criticism is provided.
19 In my view, the topic of the laboratory exercises is of little concern. Our experience involves a course centred in enzymology as outlined on Scheme 1. No doubt there are other scenarios that could be designed. It is our belief, however, that all students of biochemistry, regardless of their primary interest within the discipline, will profit from a working knowledge of the structure and function of enzymes. The single topic approach also allows one to build a series of experiments proceeding from a less complex to a more complex experimental design. The course assumes exposure to one year of didactic biochemistry (90 lectures) and previous basic laboratory experience. Experience in physical chemistry is not essential but is helpful. The first six periods of instruction after the introductory lecture provide students with some basic fundamentals of research. These include data collection and analysis including the importance of statistics, preparation of buffers, the use of microtechniques and their importance, keeping a good research notebook, designing a useful protocol, the problems
involved in assaying an enzyme and the elements of writing a good scientific paper. The fundamentals of ultraviolet-visible spectrophotometry and pH are also reviewed. After the first six periods, students begin a study of yeast alcohol dehydrogenase. Each experiment is preceded by a 15-20 minute lecture available on an audiovisual cassette. Only a minimum of didactic material is reviewed. Experiments involving the determination of first- and second-order rate constants, enzyme kinetic parameters, and recombinant DNA enzymology are simulated on a microcomputer) Audiovisual lectures, the simulated experiments on a microcomputer and tutorial assistance are provided to help the student in designing an experiment, writing the protocol and completing the experiment. The protocol must be approved by an instructor before the student has permission to initiate the laboratory work. Each student is allotted a generous but fLxed time-period in which to complete the experiment, analyze the data and write a scientific laboratory report. Laboratory reports should be expected at fixed time-periods throughout the term.
Scheme 1 One of several possible sequences of topics for a laboratory course
Introduction to Biochemical Research
I
II
III
Introduction and Objectives Basic (1) (2) (3) (4) (5) (6) (7) (8) (9)
Techniques Statistical considerations in data collection and analysis Buffers in a biochemical research laboratory Microtechniques in a biochemical research laboratory Laboratory notebooks - keeping research records Protocols - What are they? Spectrophotometry Enzyme assay - Assay of yeast alcohol dehydrogenase Handling animals - Bleeding a rabbit Report - Composition and structure
Experiments (1) Structure of yeast alcohol dehydrogenase a Number of buried and exposed sulphydryl groups by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) b Determine pseudo 1st order and 2nd order rate constant for reaction (2) Function of yeast alcohol dehydrogenase a Enzymatic assay of metabolite, NAD ÷ b Equilibrium constant of yeast alcohol dehydrogenase - effect of pH (optional) c Kinetic properties of yeast alcohol dehydrogenase; K m and NAD ÷ and Ki for NADH at infinite concentration of ethanol (3) Purification, molecular weight and antigenic properties of yeast alcohol dehydrogenase a Fractional precipitation of yeast alcohol dehydrogenase from a crude yeast extract by PEG 4000 b DEAE-cellulose chromatography c Comparative elution of yeast alcohol dehydrogenase with standard proteins from a gel permeation column using high pressure liquid chromatographic techniques d Antigenic properties of yeast alcohol dehydrogenase: (i) Ouchterlony double diffusion, and (ii) determination of antibody titre (4) Recombinant DNA enzymology a Restriction of pBR 322 and XDNA with Eco RI b Ligation of restricted pBR 322 and XDNA with T4 DNA ligase c Agarose gel electrophoresis
BIOCHEMICAL EDUCATION
10(1) 1982
No of 3 h periods recommended for completion
20 A laboratory manual is provided that outlines the objectives of each experiment. Broad guidelines are provided so that the experimental protocol can be written without prior experimentation. It is necessary to hold a discussion once a week to provide a forum for group interaction in the analysis and interpretation of data.
Description of experiments Experiment I The first experiment provides students with experience in classical chemical kinetics. The number of exposed sulphydryl groups is obtained from the change in absorption at 412 nm (e = 14,100M -1 cm-1) a after reaction of excess 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) with the sulphydryl groups. Sodium dodecyl sulphate is added to unfold the protein and expose the buried residues. Generally, all sulphydryls of yeast ADH react as exposed residues in Tris/HCl buffer, pH 8 to 9. DTNB is dissolved in water containing an equivalent amount of Na2CO3. Commercial lyophilized yeast ADH is provided to each student for this and experiment 2. Experiment 2 The kinetic parameters, Km for NAD and K i for NADH, are measured by standard spectrophotometric techniques. Data are analyzed by procedures as suggested by Wilkinson4 with the assistance of a PET microcomputer. 2 Students are given literature values for Km and Ki s as an aid in designing the experiment. Experiment3 The third experiment is more complex. It requires the application of several techniques to the partial purification of yeast ADH from a crude yeast extract. Each student is provided with 30-50 ml of a crude yeast extract prepared according to a published procedure. 6 Solid cake yeast (500 g) is crumbled into liquid N2 in a Dewar. After the yeast is frozen it and the liquid N2 are poured into a Waring Blender and blended for 4 minutes in 1 minute intervals. (The blade assembly of the blender must be dry to prevent freeze-up.) One litre of 20 mM sodium phosphate, pH 7.8, is added to the fine powder and the suspension homogenized after it has thawed. This homogenate is stirred for an additional hour and centrifuged. One-fifth volume of a 2% solution of protamine sulfate is added to the supernatant which is stirred for 20 minutes at room temperature. After centrifuging and adjusting the supernatant to pH 7 with 1:1 NH4OH, twenty-millilitre aliquots are stored in plastic scintillation vials at -20 ° for distribution to the student. The first step of the purification requires the student to determine the amount of PEG 4000 (50% w/v) required to complete a fractional precipitation. PEG 4000 concentrations ranging from 0 to 20% are appropriate. After subjecting the remaining crude yeast extract to the PEG 4000 fractionation, the precipitate is dissolved in a minimum volume of 5 mM phosphate buffer, pH 7.0. Between 10 and 20 mg of protein, estimated from the absorbance at 280 nm (assume e =0.8 ml mg -~ cm-1), at 5-10 mg/ml is applied to 8-10 ml of DEAE cellulose packed in a 10 ml disposable syringe column. This is eluted with a linear gradient of sodium phosphate composed of 25 ml of 5 mM and 25 ml of 0.15 M sodium phosphate, pH 7.5. Twenty-five 2 ml fractions are collected by hand. Protein concentration is estimated from the absorbances at 280 and 260 nm. The conductance of 0.1 ml aliquots diluted to 3 ml is measured to determine the linearity of the gradient. BIOCHEMICAL EDUCATION
1 0 ( 1 ) 1982
The peak fractions containing enzyme activity can be pooled and precipitated by dialysis against saturated ammonium sulphate. The molecular weight of commercial yeast ADH is determined by comparative elution with standard proteins on a gel permeation colum using high pressure liquid chromatographic techniques. 1 Normally 6 to 8 standard proteins are used. Experimental data for the HPLC experiment can be obtained in a 2 hour period. Data for molecular weight determination may be obtained with a calibrated gel-permeation column developed under gravity flow. Depending on the design of the experiment, this approach may be more time-consuming. Early in the term, a rabbit is bled to obtain pre-immune serum, then immunized twice, once with alcohol dehydrogenase suspended in Freund's complete adjuvant followed by a second injection 3 weeks later.* The immune serum is is collected one week after the second injection. Three students use one rabbit. Each student has an opportunity to work with the animal and perform the necessary operations. Blood is collected in Petri dishes (15 cm) and allowed to clot overnight before the serum is removed. A 'V' cut in the clot with a razor-blade increases the yield of serum. The pre-and post-immune serum is subjected to an Ouchterlony double diffusion with both the commercial yeast enzyme and the partially purified enzyme. The antibody titre is obtained from a plot of enzyme activity remaining after titration of a fixed amount of antibody with enzyme.
Experiment 4 This experiment is technically more difficult. It is completed during the last four periods after students have gained experience with microtechniques. Basically each student sets up a 30/21 reaction involving digestion separately of XDNA and pBR322 plasmid vector with a restriction endonuclease, and ligation of the XDNA fragments with the restricted vector. Electrophoresis on agarose gel and staining with ethidium bromide tests the results of the ligation and gives the students experience in interpreting gel-electrophoretic data.**
Discussion Some of the major goals of this laboratory are (1) to give students an opportunity to design their experiments, ( 2 ) t o give students experience in writing their own protocols, (3) to impress upon students the importance of using microtechniques to conserve material, (4) to provide experience in using microtechniques, (5) to give students assistance and experience in interpreting data, (6) to provide experience and constructive criticism in writing scientific research papers, ( 7 ) t o provide experience and constructive criticism in maintaining a research notebook, and (8) to impress upon the students the need to be concerned with the errors involved in an experiment. The first class period of 3 hours is devoted to introducing students to the structure of the course and the requirements for its satisfactory completion. Next we compare the format of this course to a typical research laboratory and point out that experiments in an academic research laboratory are generally designed by a student in conjunction with his/her peers and~or mentor. After the experiments are completed, *Editorial Note: Readers should be cognizant of restrictions in their respective country of doing experiments with live animals. **As soon as the material becomes available, this experiment will be done with the yeast DNA for alcohol dehydrogenase.
21 the results are shared and analyzed again in conjunction with his/her peers and/or mentor. As a result we encourage students to discuss their protocols and experimental results among themselves. The weekly group discussions provide a useful forum for discussing the data. Finally it is important to note that an experiment has no meaning unless its results are communicated to the scientific community at large. All other lecture material is provided on audiovisual cassettes. Thus it is not necessary for all students to begin each experiment at the same time. In fact, to minimize the need for multiple units of major equipment, students are encouraged to stagger the start of various experiments. Each audiovisual cassette is designed to introduce the basic elements of a technique or procedure. For instance, the cassette entitled 'The Assay of an Enzyme' covers the following topics: end point and continuous assays, direct and coupled assays, assay temperature and pH, reaction equilibrium, substrate concentration, starting the reaction, measurement of initial velocity, and specific activity. The primary disadvantage of this approach involves the need for time. First students, at least during the early part of the term, require assistance in using instruments and writing protocols. Since the structure is flexible, we have provided assistance for 4-4½ hours instead of the normal 3-hour laboratory period. As the term progresses students require less and less assistance but many need the time to complete the experiments. Students also sense a time pressure to f'mish an experiment. This often results in the presentation of a minimum number of data, sometimes without replication. It is also important to realize that everyone, including students, has a tendency to procrastinate and therefore it is important to have deadlines for reports during the tenn. Students who take this course are well satisfied. They enjoy the challenge of writing their own protocols, preparing their own reagents, working individually, and the flexibility of working at their own pace. The lectures on audiovisual cassettes and the microcomputer simulations are convenient since the background material for an experiment can be reviewed and simulated immediately prior to preparing a protocol. The main complaint revolved around the need for more time to assimilate the results of an experiment. This complaint is partially rectified by the weekly discussion period in which experimental data are discussed and analyzed. The cost of this offering is not excessive. It can be minimized by emphasizing microtechniques, using plastic cuvettes, and careful monitoring of the amount of material planned for use as indicated by the protocols. The cost can be reduced by preparing ADH from yeast. Experiment 4 may also be deleted to reduce costs without seriously jeopardizing the objectives of the laboratory experiences. Good thermostarted ultraviolet-visible recording spectrophotometers are necessary for the course indicated by Scheme 1. References 1Laidler, K J (1974) JChem Education 51, 696-700 2 Suelter, C H and Hill, D (1981) J Chem Education, in the press 3 Riddles, P W, Blakeley, R L and Zerner, B (1979) Analyt Biochem 94, 75 -81 *Wilkinson, G N (1961) Biochem J 8 0 , 324-332 SSund, H and Theorell, H (1963) in 'The Enzymes', 2nd Edition, P D Boyer, H Lardy and K Myrback, editors, Academic Press, New York, pp 25-83 6 Dethmers, J K, Ferguson-Miller, S and Margoliash, E (1979) J Biol Chem 254, 11973-11981 7 Regnier, F E and Gooding, K M (1980) Analyt Blochem 103, 1-25.
BIOCHEMICAL EDUCATION
10(1) 1982
Science Research in the Universities S L BONTING
Department of Biochemistry University of Ni]megen Ni]megen, Netherlands Introduction
In order to say anything of significance on such a broad topic it will be necessary to limit severely the points to be considered. Thus, although it would be very desirable to consider science research in the Communist countries and the Third World countries, I shall limit myself to the Western world and more particularly Western Europe. Having thus limited the topic territorially, there immediately suggest themselves for consideration the tremendous growth in science research during the sixties and early seventies and the consequences of this growth: promises full'died and unf'ilfulled, effects good and bad, societal and governmental reactions favourable and unfavourable, scientists as heroes and villains. On top of this we have the radically changed economic and social conditions in the Western world since the early seventies, which are having their repercussion on science research in the universities. These aspects will be discussed under four headings: (1) organization and f'mancing; (2) science policy; (3) further developments; and (4) the responsibility of the scientists. Organization and f'mancing Information from six representative Western European countries was obtained from a recent study by Van Rossum*: Belgium, Italy, the Netherlands, Norway, United Kingdom and Switzerland. In all of these countries university research is almost completely funded by the Government, both directly (through general university funding) and indirectly (through Research Councils); private foundations play a very minor role (2% or less), in contrast to the situation in the USA. In all cases the Ministry of Education is solely or largely responsible for university funding, but in the United Kingdom the University Grants Committee budgets, receives and distributes these funds. Typical for all these countries is the large growth from the fifties to the seventies in manpower and funds for science research in general, but particularly for that in the universities. Table 1 illustrates this for the Netherlands. Responsible for this were the favourable economic conditions in Western Europe over this period, the large increase in student enrolment with subsequent increase in staff, and probably the effect of defence spending. Table 2 shows that this rapid growth was a general phenomenon in Western Europe, even though here only figures for the latter half of this period were available. When we add to this the many European joint research installations and efforts, like CERN, Euratom, ESA (European Space Agency) and EMBL (European Molecular Biology Laboratory), we can conclude that the European science research potential now equals that of the USA. Whether this is also true for scientific productivity and number of scientific 'breakthroughs' is another question! *W. van Rossum, The Organization and Financing of {ParaJ-University Research in Western Europe: A Comparative View, 1979, 132 pp, no publisher.