530 PARt IV The Immune System in Health and Disease heterokaryons, containing nuclei from both parent cells. Ran Chromosomes dom loss of some chromosomes and subsequent cell prolifer ation yield a clone( contain a single nucleus with chromosomes fro of the fused cells such a clone is called a hybridoma. Historically, cell fusion was promoted with Sendai virus, but now it is generally done with polyethylene glycol. Normal antigen-primed B cells can be fused with cancerous plasm Normal t or B cell ancerous t or b cell cells, called myeloma cells( Figure 23-2). The hybridoma 7-10 (grows continuously thus formed continues to express the antibody genes of the normal B lymphocyte but is capable of unlimited growth, a characteristic of the myeloma cell. B-cell hybridomas that secrete antibody with a single antigenic specificity, called monoclonal antibody, in reference to its derivation from a single clone, have revolutionized not only immunology but biomedical research as well as the clinical laboratory. Chapter 4 describes the production and uses of monoclonal antibod- ies in detail (see Figures 4-21) Nucleus Nucleus of of normal T-cell hybridomas can also be obtained by fusing T lym- lymphocyte phocytes with cancerous T-cell lymphomas. Again, the result ing hybridoma continues to express the genes of the normal Tcell but acquires the immortal-growth properties of the can- cerous T lymphoma cell. Immunologists have generated a Heterokaryon number of stable hybridoma cell lines representing T-helper and T-cytotoxic lineages Random chromosomal loss Protein Biochemistry The structures and functions of many important molecules of the immune system have been determined with the tech- niques of protein biochemistry, and many of these tech- niques are in constant service in experimental immunology For example, fluorescent and radioactive labels allow immu nologists to localize and visualize molecular activities, and the ability to determine such biochemical characteristics of a (expresses some normal protein as its size, shape, and three-dimensional struct grows indefinitely like a cancer cell) provided essential information for understanding the tions of immunologically important molecules. Radiolabeling Techniques Allow Sensitive detection of antio Monoclonal or Antibodies antibody 2(12) Radioactive labels on antigen or antibody are extremely sen sitive markers for detection and quantification. There are a number of ways to introduce radioactive isotopes into pro- B- teins or peptides. For example, tyrosine residues may be labeled with radioiodine by chemical or enzymatic proce dures. These reactions attach an iodine atom to the phenol FIGURE 23-2 Production of B-cell and T-cell hybridomas by ring of the tyrosine molecule. One of the enzymatic iodin- somatic-cell hybridization. The resulting hybridomas express some tion techniques, which uses lactoperoxidase, can label pro- of the genes of the original normal B or T cell but also exhibit the teins on the plasma membrane of a live cell without labeling immortal-growth properties of the tumor cell. This procedure is used proteins in the cytoplasm, allowing the study of cell-surface to produce B-cell hybridomas that secrete monoclonal antibody and roteins without isolating them from other cell constituents. T-cell hybridomas that secrete various growth factors
heterokaryons, containing nuclei from both parent cells. Random loss of some chromosomes and subsequent cell proliferation yield a clone of cells that contain a single nucleus with chromosomes from each of the fused cells; such a clone is called a hybridoma. Historically, cell fusion was promoted with Sendai virus, but now it is generally done with polyethylene glycol. Normal antigen-primed B cells can be fused with cancerous plasma cells, called myeloma cells (Figure 23-2). The hybridoma thus formed continues to express the antibody genes of the normal B lymphocyte but is capable of unlimited growth, a characteristic of the myeloma cell. B-cell hybridomas that secrete antibody with a single antigenic specificity, called monoclonal antibody, in reference to its derivation from a single clone, have revolutionized not only immunology but biomedical research as well as the clinical laboratory. Chapter 4 describes the production and uses of monoclonal antibodies in detail (see Figures 4-21). T-cell hybridomas can also be obtained by fusing T lymphocytes with cancerous T-cell lymphomas. Again, the resulting hybridoma continues to express the genes of the normal T cell but acquires the immortal-growth properties of the cancerous T lymphoma cell. Immunologists have generated a number of stable hybridoma cell lines representing T-helper and T-cytotoxic lineages. Protein Biochemistry The structures and functions of many important molecules of the immune system have been determined with the techniques of protein biochemistry, and many of these techniques are in constant service in experimental immunology. For example, fluorescent and radioactive labels allow immunologists to localize and visualize molecular activities, and the ability to determine such biochemical characteristics of a protein as its size, shape, and three-dimensional structure has provided essential information for understanding the functions of immunologically important molecules. Radiolabeling Techniques Allow Sensitive Detection of Antigens or Antibodies Radioactive labels on antigen or antibody are extremely sensitive markers for detection and quantification. There are a number of ways to introduce radioactive isotopes into proteins or peptides. For example, tyrosine residues may be labeled with radioiodine by chemical or enzymatic procedures. These reactions attach an iodine atom to the phenol ring of the tyrosine molecule. One of the enzymatic iodination techniques, which uses lactoperoxidase, can label proteins on the plasma membrane of a live cell without labeling proteins in the cytoplasm, allowing the study of cell-surface proteins without isolating them from other cell constituents. 530 PART IV The Immune System in Health and Disease Polyethylene glycol Chromosomes Normal T or B cell (dies after 7–10 days in culture) Cancerous T or B cell (grows continuously in culture) Heterokaryon Nucleus of cancer cell Nucleus of normal lymphocyte Random chromosomal loss Hybridoma (expresses some normal B-cell or T-cell genes but grows indefinitely like a cancer cell) B-cell hybridoma T-cell hybridoma Monoclonal antibody Interleukin 2 (IL-2) FIGURE 23-2 Production of B-cell and T-cell hybridomas by somatic-cell hybridization. The resulting hybridomas express some of the genes of the original normal B or T cell but also exhibit the immortal-growth properties of the tumor cell. This procedure is used to produce B-cell hybridomas that secrete monoclonal antibody and T-cell hybridomas that secrete various growth factors
Experimental Systems CHAPTER 23 531 Radioisotopes commonly used may cause denaturation and loss of activity. A convenient la- TABLE23-4in immunology laboratories beling system has been developed which may be used in con- junction with the ELISA and ELISPOT assays described in Radiation type Autoradiography Chapter 6. This labeling technique exploits the high affinity of the reaction between the vitamin biotin and avidin, a large molecule that may be labeled with radioactive isotopes, with 6. 8 da fluorescent molecules, or with enzymes. Biotin is a small 5'Cr 27.8 da molecule(mol wt. 244)that can be coupled to an antibody (or 143da to any protein molecule) by a gentle chemical reaction that causes no loss of antibody activity. After the biotin-coupled 874da 14C5730y 235y the avidin molecule ( figure 23-3). The reaction between bio- tin and avidin is highly specific and of such high affinity that y(gamma)radiation may be detected in a solid scintillation counter. the bond between the two molecules under most assay condi B(beta)radiation is detected in a liquid scintillation counter by its ability to convert energy to photons of light in a solution containing phosphorescent tions is virtually irreversible 可dMm级 Proteins by Size and Charge Gel Electrophoresis Separates normal autoradiographic techniques. When subjected to an electric field in an electrophoresis chamber, a charged molecule will move toward the oppo- sitely charged electrode. The rate at which a charged mole- cule moves in a stable field (its electrophoretic mobility) A general radiolabeling of cell proteins may be carried out depends upon two factors specific to the molecule: one is the by growing the cells in a medium that contains one or more sign and magnitude of its net electrical charge, and the other radiolabeled amino acids. The amino acids selected for this is its size and shape. All other factors being equal, if mole application are those most resistant to metabolic modification cules are of equal size the one with higher net charge will during cell growth so that the radioactive label will appear in move faster in an applied electrical field due to the molecular the cell protein rather than in all cell constituents. Leucine seiving properties of the solid medium. It also follows that marked with"C or H, and cysteine or methionine labeled small molecules will move faster than large ones of the same withS, are the most commonly used amino acids for meta- net charge. Although there are exceptions in which the shape bolic labeling of proteins. Table 23-4 lists some properties of of a molecule may increase or decrease its frictional drag and the radioisotopes used in immunologic research cause atypical migration behavior, these general principles Biotin Labels facilitate detection Most electrophoretic separations are not conducted in of small Amounts of proteins free solution but rather in a stable supporting medium, such as a gel. The most popular in reseach laboratories is a poly- In some instances direct labeling of proteins, especially with merized and crosslinked form of acrylamide. Separation on enzymes or other large molecules, as described in Chapter 6, polyacrylamide gels, commonly referred to as Biotin active ester Labeled avidin biotinylated Ab FIGURE23-3Labeling of antibody with biotin. An antibody prepara- antibody, the bound antibody can be detected with labeled avidin.The tion is mixed with a biotin ester, which reacts with the antibody. The avidin can be radioactively labeled or linked to an enzyme that catalyzes biotin-labeled antibody can be used to detect antigens on a solid sub- a color reaction, as in ELISA procedures(see Figure 6-10) strate such as the well of a microtiter plate. After washing away unbound
A general radiolabeling of cell proteins may be carried out by growing the cells in a medium that contains one or more radiolabeled amino acids. The amino acids selected for this application are those most resistant to metabolic modification during cell growth so that the radioactive label will appear in the cell protein rather than in all cell constituents. Leucine marked with 14C or 3 H, and cysteine or methionine labeled with 35S, are the most commonly used amino acids for metabolic labeling of proteins. Table 23-4 lists some properties of the radioisotopes used in immunologic research. Biotin Labels Facilitate Detection of Small Amounts of Proteins In some instances direct labeling of proteins, especially with enzymes or other large molecules, as described in Chapter 6, may cause denaturation and loss of activity. A convenient labeling system has been developed which may be used in conjunction with the ELISA and ELISPOT assays described in Chapter 6. This labeling technique exploits the high affinity of the reaction between the vitamin biotin and avidin, a large molecule that may be labeled with radioactive isotopes, with fluorescent molecules, or with enzymes. Biotin is a small molecule (mol. wt. 244) that can be coupled to an antibody (or to any protein molecule) by a gentle chemical reaction that causes no loss of antibody activity. After the biotin-coupled antibody has reacted in the assay system, the labeled avidin is introduced and binding is measured by detecting the label on the avidin molecule (Figure 23-3). The reaction between biotin and avidin is highly specific and of such high affinity that the bond between the two molecules under most assay conditions is virtually irreversible. Gel Electrophoresis Separates Proteins by Size and Charge When subjected to an electric field in an electrophoresis chamber, a charged molecule will move toward the oppositely charged electrode. The rate at which a charged molecule moves in a stable field (its electrophoretic mobility) depends upon two factors specific to the molecule: one is the sign and magnitude of its net electrical charge, and the other is its size and shape. All other factors being equal, if molecules are of equal size the one with higher net charge will move faster in an applied electrical field due to the molecular seiving properties of the solid medium. It also follows that small molecules will move faster than large ones of the same net charge. Although there are exceptions in which the shape of a molecule may increase or decrease its frictional drag and cause atypical migration behavior, these general principles underlie all electrophoretic separations. Most electrophoretic separations are not conducted in free solution but rather in a stable supporting medium, such as a gel. The most popular in reseach laboratories is a polymerized and crosslinked form of acrylamide. Separation on polyacrylamide gels, commonly referred to as polyacrylamide Experimental Systems CHAPTER 23 531 TABLE 23-4 Radioisotopes commonly used in immunology laboratories Isotope Half-life Radiation type* Autoradiography† 125I 60.0 da + 131I 6.8 da + 51Cr 27.8 da – 32P 14.3 da + 35S 87.4 da + 14C 57.30 yrs + 3 H 12.35 yrs – * (gamma) radiation may be detected in a solid scintillation counter. (beta) radiation is detected in a liquid scintillation counter by its ability to convert energy to photons of light in a solution containing phosphorescent compounds. † Radiation may also be detected by exposure to x-ray film. 35S and 14C must be placed in direct contact with film for detection. 3 H cannot be detected by normal autoradiographic techniques. Labeled avidin Ag Ab Biotin active ester Biotinylated Ab Avidin bound to biotinylated Ab FIGURE 23-3 Labeling of antibody with biotin. An antibody preparation is mixed with a biotin ester, which reacts with the antibody. The biotin-labeled antibody can be used to detect antigens on a solid substrate such as the well of a microtiter plate. After washing away unbound antibody, the bound antibody can be detected with labeled avidin. The avidin can be radioactively labeled or linked to an enzyme that catalyzes a color reaction, as in ELISA procedures (see Figure 6-10).
532 Iv The Immune System in Health and disease To ple Buffer 30 Plastic 29 Buffer Bottom Relative mo FIGURE 23-4 Gel electrophoresis(a)A standard PAGE apparatus with cathode at the top and anode at the bottom. Samples are loaded on the top of the gel in sample wells and electrophoresis is accom- Stable plished by running a current from the cathode to the anode.(b)The obility, or distance traveled by a species during SDS. Molecules PAGE, is inversely proportional to the log of its molecular weight. The migrate to 7.0 molecular weight of a protein is readily determined by the log of its migration distance with a standard curve that plots the migration dis which their 6.0 tances of the set of standard proteins against the logs of their molecu lar weights.( c)Isoelectric focusing, or IEF, separates proteins solely by is zer 5.0 charge. Proteins are placed on a stable pH gradient and subjected to electrophoresis. Each protein migrates to its isoelectric point, the point at which its net charge is zero. Part (b) after K. Weber and M. Osbom, 975, The Proteins, 3rd ed, vol. 1, p. 179. Academic Press, gel electrophoresis(PAGE), may be used for analysis of pro- arate the components of a mixture of proteins according to teins or nudeic acids( Figure 23-4a) molecular weight. Second, because the electrophoretic mobil- In one common application, the electrophoresis of pro- ity, or distance traveled by a species during SDS-PAGE, is in- teins through a polyacrylamide gel is carried out in the pres- versely proportional to the logarithm of its molecular weight, ence of the detergent sodium dodecyl sulfate(SDS). This that distance is a measure of its molecular weight The gel is method, known as SDS-PAGE, provides a relatively simple stained with a dye that reacts with protein to visualize the and highly effective means of separating mixtures of proteins locations of the proteins. The migration distance of a protein on the basis of size. SDS is a negatively charged detergent that in question is then compared with a plot of the distances binds to protein in amounts proportional to the length of the migrated by a set of standard proteins( Figure 23-4b) protein. This binding destroys the characteristic tertiary and Another electrophoretic technique, isoelectric focusing secondary structure of the protein, transforming it into a (EF), separates proteins solely on the basis of their char negatively charged rod. A protein binds so many negatively This method is based on the fact that a molecule will move in harged SDS molecules that its own intrinsic charge becomes an electric field as long as it has a net positive or negative insignificant by comparison with the net charge of the SDs charge; molecules that bear equal numbers of positive and molecules. Therefore, treatment of a mixture of proteins with negative charges and therefore have a net charge of zero will SDS transforms them into a collection of rods whose electric not move. At most pH values, proteins(which characteristi charges are proportional to their molecular weights. This has cally bear a number of both positive and negative charges) two extremely useful consequences. First, it is possible to sep- have either a net negative or a net positive charge. However
gel electrophoresis (PAGE), may be used for analysis of proteins or nucleic acids (Figure 23-4a). In one common application, the electrophoresis of proteins through a polyacrylamide gel is carried out in the presence of the detergent sodium dodecyl sulfate (SDS). This method, known as SDS-PAGE, provides a relatively simple and highly effective means of separating mixtures of proteins on the basis of size. SDS is a negatively charged detergent that binds to protein in amounts proportional to the length of the protein. This binding destroys the characteristic tertiary and secondary structure of the protein, transforming it into a negatively charged rod. A protein binds so many negatively charged SDS molecules that its own intrinsic charge becomes insignificant by comparison with the net charge of the SDS molecules. Therefore, treatment of a mixture of proteins with SDS transforms them into a collection of rods whose electric charges are proportional to their molecular weights. This has two extremely useful consequences. First, it is possible to separate the components of a mixture of proteins according to molecular weight. Second, because the electrophoretic mobility, or distance traveled by a species during SDS-PAGE, is inversely proportional to the logarithm of its molecular weight, that distance is a measure of its molecular weight. The gel is stained with a dye that reacts with protein to visualize the locations of the proteins. The migration distance of a protein in question is then compared with a plot of the distances migrated by a set of standard proteins (Figure 23-4b). Another electrophoretic technique, isoelectric focusing (IEF), separates proteins solely on the basis of their charge. This method is based on the fact that a molecule will move in an electric field as long as it has a net positive or negative charge; molecules that bear equal numbers of positive and negative charges and therefore have a net charge of zero will not move. At most pH values, proteins (which characteristically bear a number of both positive and negative charges) have either a net negative or a net positive charge. However, 532 PART IV The Immune System in Health and Disease Apparent mass (kd) 70 10 20 30 40 50 60 0.2 0.4 0.6 0.8 1.0 Relative mobility Anode Cathode Sample wells Sample Buffer Gel Plastic frame − + + Top Mass (kd) Stable pH gradient Bottom 200 100 68 43 36 29 17 12 (a) (c) (b) Buffer Molecules migrate to position at which their net charge is zero − − − −− − + − + +− − − + pH 7.0 6.0 5.0 − 7.0 6.0 5.0 − + ++ − − − − + + + − Direction of electrophoresis FIGURE 23-4 Gel electrophoresis. (a) A standard PAGE apparatus with cathode at the top and anode at the bottom. Samples are loaded on the top of the gel in sample wells and electrophoresis is accomplished by running a current from the cathode to the anode. (b) The electrophoretic mobility, or distance traveled by a species during SDSPAGE, is inversely proportional to the log of its molecular weight. The molecular weight of a protein is readily determined by the log of its migration distance with a standard curve that plots the migration distances of the set of standard proteins against the logs of their molecular weights. (c) Isoelectric focusing, or IEF, separates proteins solely by charge. Proteins are placed on a stable pH gradient and subjected to electrophoresis. Each protein migrates to its isoelectric point, the point at which its net charge is zero. [Part (b) after K. Weber and M. Osborn, 1975, The Proteins, 3rd ed., vol. 1, p. 179. Academic Press.]
Experimental Systems CHAPTER 23 533 for each protein there is a particular pH, called its isoelectricAcidic point(pD), at which that protein has equal numbers of posi- tive and negative charges. Isoelectric focusing makes use of a gel containing substances, called carrier ampholytes, that al range themselves into a continuous pH gradient when sub- jected to an electric field. When a mixture of proteins is ap to such a gel and subjected to electrophoresis, each ein moves until it reaches that point in the gradient the pH of the gel is equal to its isoelectric point. It then stops moving because it has a net charge of zero. Isoelectric focusing is an extremely gentle and effective way of separat ing different proteins(Figure 23-4c) A method known as two-dimensional gel electrophoresis (2D gel electrophoresis) combines the advantages of SDS- PAGE and isoelectric focusing in one of the most sensitive and discriminating ways of analyzing a mixture of proteins In this method, one first subjects the mixture to isoelectric focusing on an IEf tube gel, which separates the molecules on the basis of their isoelectric points without regard to mol- ecular weight. This is the first dimension. In the next step, FIGURE 23-5 Two-dimensional gel electrophoresis of a5s-methionine one places the IEF gel lengthwise across the top of an sDs. labeled total cell proteins from murine thymocytes. These proteins polyacrylamide slab(that is, in place of the sample wells in were first subjected to isoelectric focusing(direction of migration indi- Figure 23-4a)and runs SDS-PAGE Preparatory to this step, SDSPAG arrow) and then the focused proteins were separated by all proteins have been reacted with SDS and therefore mi- SDS-PAGE(direction of migration indicated by blue arrow). The gel grate out of the IEF gel and through the SDS-PAGE slab ac- was exposed to xray film to detect the labeled proteins. [Courtesy af cording to their molecular weights. This is the second dimen- B A Osborne sion. The position of the proteins in the resulting 2D gel car be visualized in a number of ways. In the least sensitive the gel is stained with a protein-binding dye(such as Coomassie blue). If the proteins have been radiolabeled, the more sensi- silver staining is a method odphy can be used. Alternatively, microscope, the theoretical limit of resolution of the electron silver staining is a method of great sensitivity that takes ad- microscope is about 0.002 nm. If it were possible to build an vantage of the capacity of proteins to reduce silver ions to an instrument that could actually approach this limit, the elec- easily visualized deposit of metallic silver. Finally, immuno- tron microscope could readily be used to determine the blotting-blotting of proteins onto a membrane and detec- detailed atomic arrangement of biological molecules, since tion with antibody (see Figure 6-13)-can be used as a way of the constituent atoms are separated by distances of 0. 1 nm to locating the position of specific proteins on 2D gels if an ap- 0.2 nm. In practice, aberrations inherent in the operation of propriate antibody is available. Figure 23-5 shows an autora- the magnetic lenses that are used to image the electron beam diograph of a two-dimensional gel of labeled proteins from limit the resolution to about 0. 1 nm(1A). This practical limit murine thymocytes. can be reached in the examination of certain specimens, par- ticularly metals. Other considerations, however, such as X-Ray Crystallography Provides specimen preparation and contrast, limit the resolution for Structural Information biological materials to about 2 nm(20 A). To determine the arrangement of a molecule's atoms, then, we must turn to A great deal of information about the structure of cells, parts x-rays, a form of electromagnetic radiation that is readily of cells, and even molecules has been obtained by light micro- generated in wavelengths on the order of size of interatomic scopy. The microscope uses a lens to focus radiation to form distances. Even though there are no microscopes with lenses an image after it has passed through a specimen. However, a that can focus x-rays into images, x-ray crystallography can practical limitation of light microscopy is the limit of resolu- reveal molecular structure at an extraordinary level of detail. tion Radiation of a given wavelength cannot resolve struc- X-ray crystallography is based on the analysis of the diffrac tural features less than about 1/2 its wavelength. Since the tion pattern produced by the scattering of an x-ray beam as it shortest wavelength of visible light is around 400 nm, even passes though a crystal. The degree to which a particular atom the very best light microscopes have a theoretical limit of res- scatters x-rays depends upon its size. Atoms such as carbon, olution of no less than 200 nm. oxygen, or nitrogen, scatter x-rays more than do hydrogen Because of the much shorter wavelength(0.004 nm)of atoms, and larger atoms, such as iron, iodide, or mercury give the electron at the voltages normally used in the electron intense scattering X-rays are a form of electromagnetic waves:
for each protein there is a particular pH, called its isoelectric point (pI), at which that protein has equal numbers of positive and negative charges. Isoelectric focusing makes use of a gel containing substances, called carrier ampholytes, that arrange themselves into a continuous pH gradient when subjected to an electric field. When a mixture of proteins is applied to such a gel and subjected to electrophoresis, each protein moves until it reaches that point in the gradient where the pH of the gel is equal to its isoelectric point. It then stops moving because it has a net charge of zero. Isoelectric focusing is an extremely gentle and effective way of separating different proteins (Figure 23-4c). A method known as two-dimensional gel electrophoresis (2D gel electrophoresis) combines the advantages of SDSPAGE and isoelectric focusing in one of the most sensitive and discriminating ways of analyzing a mixture of proteins. In this method, one first subjects the mixture to isoelectric focusing on an IEF tube gel, which separates the molecules on the basis of their isoelectric points without regard to molecular weight. This is the first dimension. In the next step, one places the IEF gel lengthwise across the top of an SDSpolyacrylamide slab (that is, in place of the sample wells in Figure 23-4a) and runs SDS-PAGE. Preparatory to this step, all proteins have been reacted with SDS and therefore migrate out of the IEF gel and through the SDS-PAGE slab according to their molecular weights. This is the second dimension. The position of the proteins in the resulting 2D gel can be visualized in a number of ways. In the least sensitive the gel is stained with a protein-binding dye (such as Coomassie blue). If the proteins have been radiolabeled, the more sensitive method of autoradiography can be used. Alternatively, silver staining is a method of great sensitivity that takes advantage of the capacity of proteins to reduce silver ions to an easily visualized deposit of metallic silver. Finally, immunoblotting—blotting of proteins onto a membrane and detection with antibody (see Figure 6-13)—can be used as a way of locating the position of specific proteins on 2D gels if an appropriate antibody is available. Figure 23-5 shows an autoradiograph of a two-dimensional gel of labeled proteins from murine thymocytes. X-Ray Crystallography Provides Structural Information A great deal of information about the structure of cells, parts of cells, and even molecules has been obtained by light microscopy. The microscope uses a lens to focus radiation to form an image after it has passed through a specimen. However, a practical limitation of light microscopy is the limit of resolution. Radiation of a given wavelength cannot resolve structural features less than about 1/2 its wavelength. Since the shortest wavelength of visible light is around 400 nm, even the very best light microscopes have a theoretical limit of resolution of no less than 200 nm. Because of the much shorter wavelength (0.004 nm) of the electron at the voltages normally used in the electron microscope, the theoretical limit of resolution of the electron microscope is about 0.002 nm. If it were possible to build an instrument that could actually approach this limit, the electron microscope could readily be used to determine the detailed atomic arrangement of biological molecules, since the constituent atoms are separated by distances of 0.1 nm to 0.2 nm. In practice, aberrations inherent in the operation of the magnetic lenses that are used to image the electron beam limit the resolution to about 0.1 nm (1Å). This practical limit can be reached in the examination of certain specimens, particularly metals. Other considerations, however, such as specimen preparation and contrast, limit the resolution for biological materials to about 2 nm (20 Å). To determine the arrangement of a molecule’s atoms, then, we must turn to x-rays, a form of electromagnetic radiation that is readily generated in wavelengths on the order of size of interatomic distances. Even though there are no microscopes with lenses that can focus x-rays into images, x-ray crystallography can reveal molecular structure at an extraordinary level of detail. X-ray crystallography is based on the analysis of the diffraction pattern produced by the scattering of an x-ray beam as it passes though a crystal. The degree to which a particular atom scatters x-rays depends upon its size. Atoms such as carbon, oxygen, or nitrogen, scatter x-rays more than do hydrogen atoms, and larger atoms, such as iron, iodide, or mercury give intense scattering. X-rays are a form of electromagnetic waves; Experimental Systems CHAPTER 23 533 Acidic Basic FIGURE 23-5 Two-dimensional gel electrophoresis of 35S-methionine labeled total cell proteins from murine thymocytes. These proteins were first subjected to isoelectric focusing (direction of migration indicated by red arrow) and then the focused proteins were separated by SDS-PAGE (direction of migration indicated by blue arrow). The gel was exposed to x-ray film to detect the labeled proteins. [Courtesy of B. A. Osborne.]
534 PART I The Immune System in Health and Disease as the scattered waves overlap, they alternately interfere with (a) and reinforce each other. An appropriately placed detector records a pattern of spots( the diffraction pattern) whose dis- tribution and intensities are determined by the structure of the diffracting crystal. This relationship between crystal structure and diffraction pattern is the basis of x-ray crystallographic analysis. Here is an overview of the procedures used OBTAIN CRYSTAIS OF THE PROTEIN OF INTEREST To those who Diffracted beams have not experienced the frustrations of crystallizing proteins, this may seem a trivial and incidental step of an otherwise Detector (e ., film) highly sophisticated process. It is not. There is great variation from protein to protein in the conditions required to produce crystals that are of a size and geometrical formation appro- priate for x-ray diffraction analysis. For example, myoglobin (b) formed crystals over the course of several days at pH 7 in a 3 M solution of ammonium sulfate. but 1.5 m ammonium sulfate at pH 4 worked well for a human IgG1. There is no set formula that can be applied, and those who are consistently successful are persistent, determined, and, like great chefs, have a knack for making just the right"sauce SELECTION AND MOUNTING. Crystal specimens must be at least 0. 1 mm in the smallest dimension and rarely exceed a few millimeters in any dimension. Once chosen, a crystal is vested into a capillary tube along with the solution from which the crystal was grown(the mother liquor"). This keeps the crys- tal from drying and maintains its solvent content, an important consideration for maintaining the internal order of the speci men. The capillary is then mounted in the diffractionapparatus GENERATING AND RECORDING A DIFFRACTION PATTERN The precisely positioned crystal is then irradiated withx-rays of a known wavelength produced by accelerating electrons against the copper target of an x-ray tube. When the x-ray beam strikes (c) the crystal, some of it goes straight through and some is scat- tered; sensitive detectors record the position and intensity of Tyr 100H the scattered beam as a pattern of spots(Figure 23-6a, b) INTERPRETING THE DIFFRACTION PATTERN. The core of diffraction analysis is the mathematical deduction of the detailed structure that would produce the diffraction pattern observed. One must calculate to what extent the waves scat tered by each atom have combined to reinforce or cancel each other to produce the net intensity observed for each spot in Asp the array. a difficulty arises in the interpretation of complex diffraction patterns because the waves differ with respect to phase, the timing of the period between maxima and min ima. Since the pattern observed is the net result of the inter action of many waves, information about phase is critical to calculating the distribution of electron densities that is re- sponsible. The solution of this"phase problem"looms as a FIGURE 23-6 X-ray crystallography.(a)Schematic diagram of an major obstacle to the derivation of a high-resolution struc- x-ray crystallographic experiment in which an x-ray beam bombards ure of any complex molecule. the crystal and diffracted rays are detected. (b) Section of x-ray dif- The problem is solved by derivatizing the protein-mod- fraction pattern of a crystal of murine IgG2a.(c) Section from the ifying it by adding heavy atoms, such as mercury, and then electron-density map of murine IgG2a. / Part (a)from L. Stryer, 1995, obtaining crystals that have the same geometry as(are iso- Biochemistry, 4th ed. parts(b)and (c) courtesy of A McPherson./
as the scattered waves overlap, they alternately interfere with and reinforce each other. An appropriately placed detector records a pattern of spots (the diffraction pattern) whose distribution and intensities are determined by the structure of the diffracting crystal. This relationship between crystal structure and diffraction pattern is the basis of x-ray crystallographic analysis. Here is an overview of the procedures used: OBTAIN CRYSTALS OF THE PROTEIN OF INTEREST. To those who have not experienced the frustrations of crystallizing proteins, this may seem a trivial and incidental step of an otherwise highly sophisticated process. It is not. There is great variation from protein to protein in the conditions required to produce crystals that are of a size and geometrical formation appropriate for x-ray diffraction analysis. For example, myoglobin formed crystals over the course of several days at pH 7 in a 3 M solution of ammonium sulfate, but 1.5 M ammonium sulfate at pH 4 worked well for a human IgG1. There is no set formula that can be applied, and those who are consistently successful are persistent, determined, and, like great chefs, have a knack for making just the right “sauce.” SELECTION AND MOUNTING. Crystal specimens must be at least 0.1 mm in the smallest dimension and rarely exceed a few millimeters in any dimension. Once chosen, a crystal is harvested into a capillary tube along with the solution from which the crystal was grown (the “mother liquor”).This keeps the crystal from drying and maintains its solvent content, an important consideration for maintaining the internal order of the specimen.The capillary is then mounted in the diffraction apparatus. GENERATING AND RECORDING A DIFFRACTION PATTERN. The precisely positioned crystal is then irradiated with x-rays of a known wavelength produced by accelerating electrons against the copper target of an x-ray tube. When the x-ray beam strikes the crystal, some of it goes straight through and some is scattered; sensitive detectors record the position and intensity of the scattered beam as a pattern of spots (Figure 23-6a,b). INTERPRETING THE DIFFRACTION PAT TERN. The core of diffraction analysis is the mathematical deduction of the detailed structure that would produce the diffraction pattern observed. One must calculate to what extent the waves scattered by each atom have combined to reinforce or cancel each other to produce the net intensity observed for each spot in the array. A difficulty arises in the interpretation of complex diffraction patterns because the waves differ with respect to phase, the timing of the period between maxima and minima. Since the pattern observed is the net result of the interaction of many waves, information about phase is critical to calculating the distribution of electron densities that is responsible. The solution of this “phase problem” looms as a major obstacle to the derivation of a high-resolution structure of any complex molecule. The problem is solved by derivatizing the protein—modifying it by adding heavy atoms, such as mercury, and then obtaining crystals that have the same geometry as (are iso- 534 PART IV The Immune System in Health and Disease X-ray source X-ray beam Crystal Detector (e.g., film) Diffracted beams (a) (b) Tyr 100H Gly 97 Gly 96 Asp 101 Tyr 102 Tyr 100I Ala 100J Met 100K Trp 103 (c) FIGURE 23-6 X-ray crystallography. (a) Schematic diagram of an x-ray crystallographic experiment in which an x-ray beam bombards the crystal and diffracted rays are detected. (b) Section of x-ray diffraction pattern of a crystal of murine IgG2a. (c) Section from the electron-density map of murine IgG2a. [Part (a) from L. Stryer, 1995, Biochemistry, 4th ed.; parts (b) and (c) courtesy of A. McPherson.]