- Email: [email protected]
Isolation of γ-globulin (IgG) from serum: a task that encourages students to consolidate concepts on protein structure and properties
Bioche.micai
Pergamon
Education
~
Biochemical Education 26 (1998) 63-65
Isolation of ,-globulin (IgG) from serum: a task that encourages students to consolidate concepts on protein structure and properties A. I. Assis-Pandochi, A. C. Cspadaro, Y. M. Lucisano-Valim Biochemistry, Faculty of Pharrnaceutical Sciences, University of S~o Paulo, Ribeir6o Preto 14040-903, Brazil
Abstract
The isolation of 7-globulin from bovine serum by salt precipitation and fractionation through ion-exchange chromatography and gel filtration is proposed as a method to teach protein structure and properties. Students are instructed to evaluate the efficiency of the isolation procedure through SDS-PAGE analysis of protein fractions. Simple techniques to measure antigen-antibody reaction may be used to check preservation of IgG function. Questions are given to get the students to relate protein structure and physical properties and their use in protein purification and quantification. This teaching approach is efficient in identifying misunderstanding of concepts, to follow up students' development and to reinforce 'learned concepts'. © 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Work proposal
In the process of learning fundamental biochemistry, as in any other area, much extra class work is required for students before basic concepts, such as those concerning protein structure and properties, are firmly settled. However, this work is frequently only half done by students overburdened with extensive curricular programs. At the Pharmacy School of Ribeirao Preto, USP, we try to deal with these difficulties by searching for new techniques to lead students to discuss and check 'learned concepts' during class time while performing practical (bench) work. We discuss here our experience with a laboratory integration module to teach pharmacy students how to isolate and analyze a protein, reinforcing concepts about the structure and properties of these macromolecules. The laboratory integration module was initially planned for students who have taken Analytical and Organic Chemistry, Physics and Physico-chemistry, and Basic Biochemistry. In Basic Biochemistry they have studied amino acids, the various levels of protein structure related to function, carbohydrates, lipids, membranes and enzymes. Practical subjects include protein, carbohydrate and lipid characterization and determination, and enzyme kinetics.
During the first class, the project for the bench work is presented to students: the isolation of the antibody fraction from rabbit serum by the method of LucisanoValim [1] and Lucisano and Mantovani [2]. A flow chart summarizing the process is distributed and the students are divided into small groups (3-5 students) to carry out the work. General discussions follow after each step of the process and, at the end, a test is applied or students are asked to write up a report of their results and conclusions. Figure 1 shows the flow chart developed for the project presented to the students. When they receive it and see the material available over the bench, initially they feel lost and are prompted to ask questions before starting the work. Examples of these questions are given in Table 1 for steps 1 and 2 to illustrate the teaching procedure. Aspects to be discussed in the six steps are summarized in Table 2. Additional aspects to be discussed obviously depend on the circumstances. In step 5 each group constructs the elution profile by gel filtration (Fig. 2) and ion exchange chromatography (Fig. 3) based on absorbance at 280 nm and on the 'spot test' [3] using Coomassie Brilliant Blue staining. The convenience of choosing between these two methods is
0307-4412/98/$19.00+ 0.00 © 1998 IUBMB. Published by ElsevierScience Ltd. All rights reserved. P l h S0307-4412(97)00121-0
64
A. I. Assis-Pandochi et al./Biochemical Education 26 (1998) 63-65
Step Number 1
Table 2 Summary of topics to be discussed
Prog~nre
Collection of rabbit serum
$ Precipitation with ammonium sulfate (40% saturation)
$
Dialysis and protein quantification
$ Chromatography
/ *Sephadex G-200
\ DEAE-Cellulose
/
\
Elution profile (A280 nm and "spot test")
$
6
SDS-PAGE analysis
Protein salting in and salting out Protein fractionation by salt. Adequacy of the use of salt solutions or solid salts. Calculation of volume of salt-saturated solutions to be used Use of nomograms to calculate the mass of salt to add in each condition Protein dialysisAlternative procedures (desaiting on Sephadex G-25) Concentration of protein solutions Light absorption by protein at 280 nm and at 220 nm (peptide bond) Protein quantification Protein staining (peptide bond, aromatic amino acids) Protein charge x pH Protein shape and charge-effect on molecular sieving SDS-PAGE protein electrophoresis Principles and concepts Effect of SDS on proteins
Depending on the interest, a rabbit immune serum such as anti-bovine serum albumin can be used as a source of y-globulin. Protein can be quantified before and after each step, permitting the determination of purified y-globulin yield, and simple immunochemical techniques (immunodifusion, immunoelectrophoresis) used to show the preservation of protein function (and structure) throughout the process.
Fig. 1. Flow chart for 7-globulin isolation from serum. *Sepharose CL-4B can be alternativelyused (see text for comments).
3. Conclusions discussed. Use of Sephadex G-200 was indicated as firstly described [1] and is suitable for one goal chromatography. For a multi-use purpose Sepharose CL-4B is a better choice. In this case a three-fraction elution profile will be shown: a throughout pure IgM, a intermediary fraction containing IgG and the last fraction containing IgG and other contaminants such as albumin. Based on S D S - P A G E analysis [4] (Fig. 4) after step 6, the efficiency of the two chromatographic techniques for IgG purification are compared. The two bands pattern observed in the y-globulin fraction purified by DEAEcellulose chromatography is due to differential glycosylation of molecules [5]. Immunoelectrophoresis of this material against anti-rabbit y-globulin antibody shows only one typical precipitin line as expected. The use of the same procedure to isolate IgM is also discussed.
In our experience this teaching approach has been very efficient in identifying misunderstanding of concepts, to follow up student development, and to reinforce fundamental concepts. Moreover, the students are stimulated to recall concepts of physics and chemistry, and to associate them with protein properties and behavior in solutions in order to carry out their practical work.
1,2 1
A i
I i
O,S
0,4
Table 1 Examples of questions for discussion in steps 1 and 2 What serum volume should be used? (What is the 7-globulin concentration in normal serum?) Why not use plasma instead serum? Does this protein require low temperatures to maintain its function? How does it function? Is it necessary to add protease inhibitors? Why? Why proteins precipitate in salt solutions? Does ]'-globulin precipitate in 40% salt concentration? Gentle gradual addition of salt is required. Why?
0,8
0,2
0
;...i 0
.... 10
;..---, .... 20 30
, ....
40
F ~
"spot testR
, ....
, ....
60 60 nufllber
, .... 1'0
,
80
....
' .... 90 100
Ioooooooooeooeeeee®ol
Fig. 2. Elution profile (Az~, nm and spot test) after gel filtration (steps 4 and 5, Fig. 1). Sample: 0.5 ml ( 15 mg of protein); column: 2.6 x 40 cm; buffer: 0.1 M borate pH 8.0; flow rate: 5 ml/h; fraction volume: 1.0 ml; (peak h IgM throughout).
A. L Assis-Pandochi et aL/Biochemical Education 26 (1998) 63-65
65
1,5 1,26
l
i o:ir I 0-0
Na
CI1M
0,25
"sDottest"
•
/
70
80
90
100
loooooo ..ooo..... ol
Fig. 3. Elution profile (A2~,~nmand spot test) after DEAE-cellulose chromatography (steps 4 and 5, Fig. 1). Sample: 5.0 ml (150 mg of protein); column: 2.6×40cm (212ml); buffers: 0.02M phosphate pH 7.4 (equilibrium) and 0.02 Mphosphate pH 7.4 containing 1~ NaC1 (applied as indicated in the figure); flow rate 60 ml/h; fraction volume: 5.0 ml; (peak 1 is IgG throughout). T h e m e t h o d used here for isolation of 7-globulin is simple and efficient, and the protein activity is easily preserved, giving the students the feeling of a job welldone. D e p e n d i n g on the student level, m o r e general questions can be asked about research procedures, such as choice of animal species and source of material, alternative procedures available for protein fractionation, etc. A t the end a report can be p r e p a r e d by the students, representing an initial training in scientific writing. A video is being prepare d to provide a general overview of this laboratory integration module, as well as m o r e detailed discussion of each step for a better teaching approach.
Acknowledgements T h e authors gratefully~ acknowledge A n a Cristina Morseli Polizello and A n a Elisa Caleiro Seixas Azzolini
Fig. 4. SDS-PAGE analysis of samples after DEAE-cellulose and Sephadex G-200 fractionation (step 6, Fig. 1). 1,6: HMW protein standards; 2: fractions 34-45 from DEAE-cellulose; 3: fractions 65-85 from DEAE-cellulose; 4: fractions 44-54 from Sephadex G-200; 5: fractions 72-90 from Sephadex G-200; 7: LMW protein standards. The two bands pattern represent different degrees of glycosylation of the ~/-globulin molecules.
for technical assistance and Maria Regina de P R a p h a loski for typing the manuscript.
References [1] Y.M. Lucisano-Valim, Doctoral Thesis, Faculdade de Medicina de Ribeir~.o Preto-USP, 1983. [2] Y.M. Lucisano, B. Mantovani, J. Immunol. 132 (1984) 2015- 2020. [3] A. Esen, Anal. Biochem. 89 (1978) 264-273. [4] U.K. Laemmli, Nature 227 (1970) 680. [5] M. Brfiggemann, G.T. Williams, C.I. Bindon, M.R. Clark, M.R. Walker, R. Jefferis, H. Waldmann, M.S. Neuberger, J. Exp. Med. 166 (1987) 1351-1361.
Pergamon
Education
~
Biochemical Education 26 (1998) 63-65
Isolation of ,-globulin (IgG) from serum: a task that encourages students to consolidate concepts on protein structure and properties A. I. Assis-Pandochi, A. C. Cspadaro, Y. M. Lucisano-Valim Biochemistry, Faculty of Pharrnaceutical Sciences, University of S~o Paulo, Ribeir6o Preto 14040-903, Brazil
Abstract
The isolation of 7-globulin from bovine serum by salt precipitation and fractionation through ion-exchange chromatography and gel filtration is proposed as a method to teach protein structure and properties. Students are instructed to evaluate the efficiency of the isolation procedure through SDS-PAGE analysis of protein fractions. Simple techniques to measure antigen-antibody reaction may be used to check preservation of IgG function. Questions are given to get the students to relate protein structure and physical properties and their use in protein purification and quantification. This teaching approach is efficient in identifying misunderstanding of concepts, to follow up students' development and to reinforce 'learned concepts'. © 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Work proposal
In the process of learning fundamental biochemistry, as in any other area, much extra class work is required for students before basic concepts, such as those concerning protein structure and properties, are firmly settled. However, this work is frequently only half done by students overburdened with extensive curricular programs. At the Pharmacy School of Ribeirao Preto, USP, we try to deal with these difficulties by searching for new techniques to lead students to discuss and check 'learned concepts' during class time while performing practical (bench) work. We discuss here our experience with a laboratory integration module to teach pharmacy students how to isolate and analyze a protein, reinforcing concepts about the structure and properties of these macromolecules. The laboratory integration module was initially planned for students who have taken Analytical and Organic Chemistry, Physics and Physico-chemistry, and Basic Biochemistry. In Basic Biochemistry they have studied amino acids, the various levels of protein structure related to function, carbohydrates, lipids, membranes and enzymes. Practical subjects include protein, carbohydrate and lipid characterization and determination, and enzyme kinetics.
During the first class, the project for the bench work is presented to students: the isolation of the antibody fraction from rabbit serum by the method of LucisanoValim [1] and Lucisano and Mantovani [2]. A flow chart summarizing the process is distributed and the students are divided into small groups (3-5 students) to carry out the work. General discussions follow after each step of the process and, at the end, a test is applied or students are asked to write up a report of their results and conclusions. Figure 1 shows the flow chart developed for the project presented to the students. When they receive it and see the material available over the bench, initially they feel lost and are prompted to ask questions before starting the work. Examples of these questions are given in Table 1 for steps 1 and 2 to illustrate the teaching procedure. Aspects to be discussed in the six steps are summarized in Table 2. Additional aspects to be discussed obviously depend on the circumstances. In step 5 each group constructs the elution profile by gel filtration (Fig. 2) and ion exchange chromatography (Fig. 3) based on absorbance at 280 nm and on the 'spot test' [3] using Coomassie Brilliant Blue staining. The convenience of choosing between these two methods is
0307-4412/98/$19.00+ 0.00 © 1998 IUBMB. Published by ElsevierScience Ltd. All rights reserved. P l h S0307-4412(97)00121-0
64
A. I. Assis-Pandochi et al./Biochemical Education 26 (1998) 63-65
Step Number 1
Table 2 Summary of topics to be discussed
Prog~nre
Collection of rabbit serum
$ Precipitation with ammonium sulfate (40% saturation)
$
Dialysis and protein quantification
$ Chromatography
/ *Sephadex G-200
\ DEAE-Cellulose
/
\
Elution profile (A280 nm and "spot test")
$
6
SDS-PAGE analysis
Protein salting in and salting out Protein fractionation by salt. Adequacy of the use of salt solutions or solid salts. Calculation of volume of salt-saturated solutions to be used Use of nomograms to calculate the mass of salt to add in each condition Protein dialysisAlternative procedures (desaiting on Sephadex G-25) Concentration of protein solutions Light absorption by protein at 280 nm and at 220 nm (peptide bond) Protein quantification Protein staining (peptide bond, aromatic amino acids) Protein charge x pH Protein shape and charge-effect on molecular sieving SDS-PAGE protein electrophoresis Principles and concepts Effect of SDS on proteins
Depending on the interest, a rabbit immune serum such as anti-bovine serum albumin can be used as a source of y-globulin. Protein can be quantified before and after each step, permitting the determination of purified y-globulin yield, and simple immunochemical techniques (immunodifusion, immunoelectrophoresis) used to show the preservation of protein function (and structure) throughout the process.
Fig. 1. Flow chart for 7-globulin isolation from serum. *Sepharose CL-4B can be alternativelyused (see text for comments).
3. Conclusions discussed. Use of Sephadex G-200 was indicated as firstly described [1] and is suitable for one goal chromatography. For a multi-use purpose Sepharose CL-4B is a better choice. In this case a three-fraction elution profile will be shown: a throughout pure IgM, a intermediary fraction containing IgG and the last fraction containing IgG and other contaminants such as albumin. Based on S D S - P A G E analysis [4] (Fig. 4) after step 6, the efficiency of the two chromatographic techniques for IgG purification are compared. The two bands pattern observed in the y-globulin fraction purified by DEAEcellulose chromatography is due to differential glycosylation of molecules [5]. Immunoelectrophoresis of this material against anti-rabbit y-globulin antibody shows only one typical precipitin line as expected. The use of the same procedure to isolate IgM is also discussed.
In our experience this teaching approach has been very efficient in identifying misunderstanding of concepts, to follow up student development, and to reinforce fundamental concepts. Moreover, the students are stimulated to recall concepts of physics and chemistry, and to associate them with protein properties and behavior in solutions in order to carry out their practical work.
1,2 1
A i
I i
O,S
0,4
Table 1 Examples of questions for discussion in steps 1 and 2 What serum volume should be used? (What is the 7-globulin concentration in normal serum?) Why not use plasma instead serum? Does this protein require low temperatures to maintain its function? How does it function? Is it necessary to add protease inhibitors? Why? Why proteins precipitate in salt solutions? Does ]'-globulin precipitate in 40% salt concentration? Gentle gradual addition of salt is required. Why?
0,8
0,2
0
;...i 0
.... 10
;..---, .... 20 30
, ....
40
F ~
"spot testR
, ....
, ....
60 60 nufllber
, .... 1'0
,
80
....
' .... 90 100
Ioooooooooeooeeeee®ol
Fig. 2. Elution profile (Az~, nm and spot test) after gel filtration (steps 4 and 5, Fig. 1). Sample: 0.5 ml ( 15 mg of protein); column: 2.6 x 40 cm; buffer: 0.1 M borate pH 8.0; flow rate: 5 ml/h; fraction volume: 1.0 ml; (peak h IgM throughout).
A. L Assis-Pandochi et aL/Biochemical Education 26 (1998) 63-65
65
1,5 1,26
l
i o:ir I 0-0
Na
CI1M
0,25
"sDottest"
•
/
70
80
90
100
loooooo ..ooo..... ol
Fig. 3. Elution profile (A2~,~nmand spot test) after DEAE-cellulose chromatography (steps 4 and 5, Fig. 1). Sample: 5.0 ml (150 mg of protein); column: 2.6×40cm (212ml); buffers: 0.02M phosphate pH 7.4 (equilibrium) and 0.02 Mphosphate pH 7.4 containing 1~ NaC1 (applied as indicated in the figure); flow rate 60 ml/h; fraction volume: 5.0 ml; (peak 1 is IgG throughout). T h e m e t h o d used here for isolation of 7-globulin is simple and efficient, and the protein activity is easily preserved, giving the students the feeling of a job welldone. D e p e n d i n g on the student level, m o r e general questions can be asked about research procedures, such as choice of animal species and source of material, alternative procedures available for protein fractionation, etc. A t the end a report can be p r e p a r e d by the students, representing an initial training in scientific writing. A video is being prepare d to provide a general overview of this laboratory integration module, as well as m o r e detailed discussion of each step for a better teaching approach.
Acknowledgements T h e authors gratefully~ acknowledge A n a Cristina Morseli Polizello and A n a Elisa Caleiro Seixas Azzolini
Fig. 4. SDS-PAGE analysis of samples after DEAE-cellulose and Sephadex G-200 fractionation (step 6, Fig. 1). 1,6: HMW protein standards; 2: fractions 34-45 from DEAE-cellulose; 3: fractions 65-85 from DEAE-cellulose; 4: fractions 44-54 from Sephadex G-200; 5: fractions 72-90 from Sephadex G-200; 7: LMW protein standards. The two bands pattern represent different degrees of glycosylation of the ~/-globulin molecules.
for technical assistance and Maria Regina de P R a p h a loski for typing the manuscript.
References [1] Y.M. Lucisano-Valim, Doctoral Thesis, Faculdade de Medicina de Ribeir~.o Preto-USP, 1983. [2] Y.M. Lucisano, B. Mantovani, J. Immunol. 132 (1984) 2015- 2020. [3] A. Esen, Anal. Biochem. 89 (1978) 264-273. [4] U.K. Laemmli, Nature 227 (1970) 680. [5] M. Brfiggemann, G.T. Williams, C.I. Bindon, M.R. Clark, M.R. Walker, R. Jefferis, H. Waldmann, M.S. Neuberger, J. Exp. Med. 166 (1987) 1351-1361.