SPECIAL REPORT
5
Ι^ϋ ^ UIMOra^MSM? A valuable technique for purifying enzymes, studying cell interactions, and exploring hormone receptors may also have potential in treating disease Indu Parikh and Pedro Cuatrecasas, Wellcome Research Laboratories Since its inception in 1968, affinity chromatography has helped solve an increasing number of problems in the biomedical sciences. By this technique, a large number of enzymes have been purified to homogeneity. Many previously unrecognized isoenzymes have been discovered. And the method has been used to solve problems as diverse as the mechanisms of enzyme action, the nature of cell interactions, the mode of immunological and hormone-receptor actions, and the treatment of diseases. The basic concepts of so-called "biospecific adsorption" were known to Emil Starkenstein at the Deutsche Universitât in Prague as early as 1910. However, they generally were not perceived as potentially useful laboratory tools until their formal reintroduction and christening as "affinity chromatography" in 1968 by Pedro Cuatrecasas, Meir Wilchek, and Christian B. Anfinsen, then all at the National Institutes of Health. The impetus for that development was research on the chemistry of the active site of the model enzyme staphylococcal nuclease by affinity labeling with selective inhibitors. The reasoning is simple. Certain chemically reactive substrate inhibitors, by virtue of their intrinsic affinity and specificity, can find their way to the active site of a protein and then attach themselves there reversibly. Similarly, an enzyme can find its way and attach itself, reversibly through its active site, to a substrate inhibitor that is irreversibly attached to a solid support. These concepts were applied successfully to the purification of several model enzymes, and methods were developed to expand their usefulness. By 1970, Cuatrecasas had worked out in some detail a basic set of practical procedures and simple chemical strategies for derivatizing and attaching enzyme inhibitors and other ligands to solid supports for use as specific adsorbents.
Since then, other scientists have applied this technique in many areas of biochemistry and biology. The purification of proteins and nucleic acids by conventional chromatographic methods usually involves tedious, multistep procedures that give low yields and are often unsuccessful. These procedures rely
A variety of ligands are used in affinity chromatography to purify proteins Llgand
Coenzyme A, adenosine monophosphate Coenzyme B12
Flavin Folate
Proteins purified
Succinic thiokinase, phosphofructokinase Vitamin B12-binding proteins, transcobalamin I and II, methylmalonyl-CoA mutase Flavin-binding protein, luciferase Dihydrofolate reductase, folatebinding protein Various dehydrogenases
Nicotinamide adenine dinucleotide, adenosine mono- and diphosphates Pyridoxal phosphate Tyrosine aminotransferase, aspartate aminotransferase, glutamate oxaloacetate transaminase Nucleotide phosphates Various nucleotide mono-, Various kinases, aldolase, citrate di-, and triphosphates synthetase, ribonuclease reductase, sialyltransferase, galactosyltransferase, glycogen synthetase Various glycoproteins Kinases, dehydrogenases, ribonuclease, serum albumin, interferon RNA, DNA, polyadenylic Polymerases, mRNA, cDNA and polythymidylic acids
August 26, 1985 C&EN 17
Special Report
Affinity chromatography involves reversible attachment of a protein to an inhibitor Solid support, such as an agarose bead / Enzyme A inhibitor / (a ligand)
( VT*4>
ΣΙ 3 3
D
Mixture of proteins
Spacer arm, such as hexamethylenediamine
f
Enzyme A
Attachment of desired enzyme
WMMAAAMT>
~)
J
c Γ-aaD Washing buffer
(
VMM\\AWWP>
Removal of contaminating proteins
c
Elution buffer
Ι^Σ ( V
Vwwvwvwvf^> J ^
Recovery of purified enzyme A
Reusable affinity matrix The substrate inhibitor (in the case above, inhibitor of enzyme A), which is irreversibly attached to a solid support via a spacer arm, selectively adsorbs an enzyme (enzyme A) from a mixture of proteins. After the unadsorbed contaminating proteins are washed away, the enzyme can be eluted and recovered from the affinity matrix
zyme in a crude mixture will interact with and bind (adsorb) to the immobilized ligand. After unadsorbed, contaminating proteins are removed, the specifically bound enzyme is eluted from the solid support by dis rupting the enzyme-ligand interaction by one of several procedures, such as changing the ionic strength or pH of the elution buffer. Thus, selective inhibitors, cofactors, and even substrates can be used to isolate specific enzymes by affinity chromatography. Immobilized drugs, vitamins, peptides, and hormones also may be used to isolate corresponding receptors or transport proteins. Immobilized proteins can serve to isolate other (complementary or interacting) proteins. In addition, particulate biological specimens, such as cell membranes and even intact cells bearing specific re ceptors, can be separated. Specific organelles and intact cells also have been isolated with immobilized lectins or specific antibodies. Polynucleotides, antigens, anti bodies, viruses, and mutant enzyme forms have been purified by use of the same principles. Affinity chromatography has other important and exciting uses. Immobilized ligands, substrates, or en zymes can be used to study the nature of biological in teractions, the mechanism and kinetic constants of en zyme reactions, the subunit interactions of oligomeric proteins, and the mechanisms of cell-surface triggering of hormonal or immunological responses. The method also can be used to concentrate proteins present in minute quantities in biological fluids and to quantitate subnanogram amounts of such proteins. In recent years, this technique has found an expanding range of appli cations in analytical biochemistry. Moreover, a number of companies are using affinity chromatography for industrial-scale purification of proteins. For example, Wellcome Biotechnology Ltd. in London has been pu rifying interferon by this method since the late 1970s. The rapid growth of this field is evidenced by the recent proliferation of adaptations of the basic method, many of which now have their own nomenclature. For example, many potentially valuable uses have been suggested in medical diagnosis and in therapeutics ("affinity therapeutics") for a variety of diseases.
Solid supports strictly on the size, electrical charge, or other physical and chemical differences of the components of a mix ture. Often, these differences are not great enough to permit effective separation. In contrast, purification by affinity chromatography is based on a unique and fundamental biological prop erty of biological macromolecules: their selective, high-affinity recognition of, and reversible interaction with, other molecules. Like enzymes, most biological macromolecules have a substrate or functional binding site for a specific ligand or an effector molecule, which itself may be another protein. And most biological interactions involve a reversible step in their sequence. In practice, a specific ligand, such as an enzyme in hibitor, is covalently attached to a solid support, such as agarose or even to the inner surface of a glass beaker. Usually, agarose with a covalently attached ligand is packed into a chromatographic column. A specific en 18
August 26, 1985 C&EN
A wide variety of materials have been used as solid supports or matrices in affinity chromatography. An ideal matrix would have many of the properties common to an ideal support for gel filtration. The matrix should be mechanically strong, sufficiently hydrophilic to avoid the nonspecific binding of proteins, and stable to most water-compatible organic solvents. For most purposes, the matrix should be porous to allow easy diffusion of large protein molecules. A porous matrix not only per mits a high degree of ligand substitution but is more accessible to larger protein molecules. The most useful matrices are polysaccharide based, although polyamides and microporous glass beads often have been used successfully. For various reasons, poly styrene and polyurethanes generally have not been re garded as appropriate matrices in purifying proteins by affinity chromatography. The most popular matrix for affinity chromatography
Several reactions can be used for coupling ligands to derivatized agarose
α Nucleic acid
r\
Nucleic acid
NH(CH2)nNHCO(CH2)2CONHR
RNhL
NH(CH2)n NH CO (CH2)2COOH
f
VNH(CH2)nNHCOR
-NO, NH(CH2)nNH2
—
Ο
Agarose (CNBr-activated)
N-OCOCH.Br
f
ι
VNH(CH2)nNHCO-Hf~\-N02 Reduction
Protein NH(CH2)nNHCOCH2Br n
RNH2
S~\ f
I
or
R^Q>-OH (
Q
NH(CH2)nNHCOCH(CH2)2SH ι NHCOCH.
/ΓΛ |~NH(CH 2 ) n NHCOH^VNH 2 Diazotization
^NH(CH2)nNHCOH^VNaN
Alkyl or aryl derivatives
Thioester or thioether derivatives
•OH Rs
or
Azo derivatives
Most of the chemical reactions for coupling ligands with various functional groups to derivatized agarose are performed under very mild conditions and often in predominantly aqueous media
is beaded agarose, a specially purified form of the nat urally occurring polysaccharide agar. The porosity of agarose results from the polymer's ability to form a highly hydrated open meshwork structure. Agarose is quite stable to aqueous solutions of both intermediate pH and ionic strength. It is unstable, however, to tem peratures above 40 °C and to certain organic solvents. The porous meshwork of agarose can be strengthened greatly by crosslinking it with epichlorohydrin. Acti vation of agarose with cyanogen bromide also induces substantial crosslinking and stability. Magnetic metal particles coated with a water-insoluble polymer were first introduced as a novel matrix in 1976 by Martin F. Chaplin and John F. Kennedy of the Uni versity of Birmingham, England. Magnetic beads can be separated easily from biological homogenates or fluids containing colloids and other particulate matter simply
by placing the reaction container on a magnet. The re sulting rapid retrieval is not possible by conventional methods, such as centrifugation. The magnetic proce dure is particularly useful in separating labile proteins from the hostile proteolytic environment often en countered in tissue homogenates. This inherently attractive idea has been picked up and modified by other investigators. Magnetic polymers containing ultramicroscopic ferrite particles (100 Â) embedded in agarose have been used recently by Klaus Mosbach and coworkers of the University of Lund, Sweden, in affinity chromatography. Appropriately derivatized magnetic beads also have been used in solid-phase radioimmunoassays, eliminating the need for time-consuming multiple centrifugation steps. Very often, successful use of affinity chromatography depends on, among other factors, the selection of the August 26, 1985 C&EN
19
Special Report proper chemical reactions for immobilizing the ligand. The presence of residual ionic charges on the derivatized matrix may introduce complications during purification. The relative instability of certain chemical bonds formed during a coupling or activation reaction may result in the slow release of ligand, thus reducing the capacity of an affinity adsorbent. Despite the large number of methods available for activating agarose, the original cyanogen bromide method is still the most popular. Use of cyanogen bro mide for activating Sephadex, a synthetic polysaccharide matrix, for enzyme immobilization was introduced in 1967 by Jerker O. Porath and coworkers at the University of Uppsala, Sweden.
Polysaccharides and polyamides frequently serve as matrices
Spacer-arm concept
OH ' OH -D-galactose-3,6-anhydro-L-galact03e-D-galactose Cellulose OH
CH,OH
CH2OH
OH
OH
CH.OH
Crosslinked dextran (Sephadex) -0-CH2
X—ο
λ—ov
Crosslinked polyacrylamide CH 2 -CH-CH 9 -CH— CH.-CH — CH,—CH-
I 1 ι CO CO CO CO ι I I NH NH. NH NH, ι CH 2 CH 2 NH22 NH 2 NH NH I i I I CO CO CO CO I J J H~ - C H r - CJH -CH 9 —CH — C H 9 - C H - C H 0 — C
20
August 26, 1985 C&EN
Soon afterwards, Cuatrecasas and colleagues extended the method to agarose for immobilizing small ligands during their early work on affinity chromatography. Agarose is activated by cyanogen bromide in aqueous suspensions at a controlled pH. The activated agarose readily couples with nucleophilic substances, such as primary and secondary amines. The relative instability of the intermediates at neutral and basic pH necessitates rapid treatment of the activated agarose with the amino-ligand to be coupled. Meir Wilchek and co workers (now at Weizmann Institute of Science in Rehovot, Israel) have recently shown that the major products of the activation are cyanate esters and imidocarbonates. Sometimes, it is necessary to immobilize a ligand by an alternate method. Many other chemical reactions can be used to attach ligands to insoluble supports. When designing an affinity adsorbent, consideration must be given to the effect of immobilization on both the affinity and specificity of the ligand, as well as the direct effect of the matrix itself on the system. The matrix may sterically hinder the ligand's accessibility to the protein's active site. Very early in the development of affinity chromatography, the concept of a spacer arm was in troduced by Cuatrecasas and coworkers in an attempt to solve this problem. They found that a short alkyl chain inserted between the ligand and the matrix reduces or eliminates the steric influence of the matrix. Among the frequently used spacer arms is hexamethylenediamine. One of the amino groups of the spacer is attached to the matrix; the other amino group is used to couple the ligand, usually through an amide bond. Many simple reactions can be performed to con vert the terminal amino group of the spacer to obtain a variety of alternative functional groups for ligand at tachment. Hexamethylenediamine spacer arms, how ever, generate strong nonspecific binding interactions because of their hydrophobic nature. Spacer arms with minimal nonspecific binding may be obtained by interposing an ether or a secondary amine group between two short alkyl chains. Commer cially available 3,3-diaminodipropylamine and a variety of macromolecular spacers were developed by Cuatre casas and associates to help minimize the nonspecific and hydrophobic adsorption of proteins. In certain cases, however, hydrophobic spacer arms may provide regions of additional interactions with the protein to be purified and thus may be desirable. Unfortunately, no exact theory exists to predict the effects of the length, polarity, rigidity, and structure of a spacer arm on the binding of a ligand. Generalizations can be made only from em pirical observations of specific systems. In our lab, experience in isolating estrogen receptors, as well as other high-affinity membrane-localized hor mone receptors, has shown that virtually all systems using cyanogen bromide for coupling result in a sig nificant degree of solubilization or leakage of the im mobilized ligand. Leakage can be quite undesirable in high-affinity systems if the affinity of the free ligand is very much greater than that of the immobilized ligand.
represents an apparent contradic tion to the idea of absolute speci ficity described earlier. Unlike substrates and inhibitors that are highly specific for given enzymes, Ο some cofactors or coenzymes are li -0-C--NH 2 common for whole classes of en zymes. An immobilized cofactor then may be used to purify each -OH member of a given class of enzymes Carbamates if proper selective elution condi (minor and inert product) tions can be devised. Polysaccharide In 1971, Peter D. G. Dean and as matrix sociates, then at the University of Liverpool, England, introduced OH J-O-CSN general-ligand affinity chroma CNBr C=NH tography and purified a variety of related dehydrogenases using agarose-immobilized adenosine Imidocarbonates monophosphate. This ligand is a part of the cofactor nicotinamide adenine dinucleotide. The method RNH was later used extensively by Mosbach and associates at the Univer NH sity of Lund. Individual dehydro II -O-C-NHR genases can be selectively eluted from the affinity column by an C=NR elution buffer containing increas -OH ing amounts of the cofactor alone or Major product Major product formed in of mixtures of the cofactor and the formed In agarose dextrans corresponding enzyme substrate. R ~ Alfcyl or aryf group Nathan O. Kaplan and coworkers at the University of California, San Diego, have used a series of adsor Cyanate esters are formed predominantly in agarose, whereas imidocarbonates bents with various analogs of are formed in Sephadex as reactive intermediates. Such reactive intermediates adenosine triphosphate to study the are produced to couple alkyl or aryl amines readily to polysaccharide supports elution profiles of various protein kinases. Other immobilized cofac tors and coenzymes, such as coen zyme A, folic acid, flavin, thiamine, and pyridoxal, have been used to purify corresponding Such leakage can be reduced drastically by multipoint classes of enzymes. attachment of the ligand to the matrix. In this approach, An extension of general-ligand affinity chromatog branched copolymers of lysine (backbone) and alanine raphy makes use of a variety of immobilized triazine (side chains) or even denatured bovine serum albumin dyes of different shades and colors as group-specific can be used as polyfunctional anchoring spacers, which adsorbents. The supposedly inert Dextran Blue, intro greatly reduce ligand leakage owing to their multipoint duced initially in the early 1970s for calibrating gelattachment. The probability of a spontaneous release of permeation chromatographic columns, was found to the ligand decreases geometrically with the number of bind selectively to certain kinases and dehydrogenases. attachment points. For example, if a ligand attached Cibacron Blue, the triazine-based chromophore of through a single point is released with a probability of Dextran Blue, inhibits the binding of adenosine tri 1000 ppm (0.1%), a ligand attached at two points (by the phosphate (but not of other substrates) to a number of same chemical bond) will be released with a probability kinases. This dye attaches itself specifically to the nu of 1 ppm (0.0001%). cleotide binding site of these enzymes. However, ac As their experience and insight have increased, sci cording to recent studies, Cibacron Blue has a broad entists have developed various ingenious modifications range of specificity for proteins, including some without of affinity chromatography. With every modification of a nucleotide binding site. Nevertheless, immobilized this technique, special consideration is given to the na Cibacron Blue F3G-A dye has been used quite success ture and kind of ligand, spacer arm, adsorption method, fully to purify various dehydrogenases, kinases, CoAand elution procedure used for a selected class of bio and polynucleotide-dependent enzymes, restriction logical macromolecules. endonucleases, polynucleotide synthetases, serum al One of the broadest subclasses of affinity chroma bumin, complement factors, and human interferons. tography, general-ligand affinity chromatography,
Cyanogen bromide is used most commonly to activate polysaccharide solid supports
August 26, 1985 C&EN
21
Special Report
Ligands are attached to supports using spacers to reduce steric hindrance
Denatured albumin on agarose
Copolymer of poly-Uysine and poly-DL-alanine on agarose In the top diagram, denatured albumin is covalently attached at two or more points on agarose by the cyanogen bromide reaction. The free e-amino groups (black dots) of the immobilized albumin can be used for covalent attachment of a ligand. The bottom diagram shows the multipoint attachment of a copolymer of poly-L-lysine (red) and poly-DL-alanine (blue). The black dots represent terminal amino groups to which a ligand may be attached covalently. Such macromolecular spacers allow large distances (50 to 150 Â) between the matrix and a ligand to help reduce steric hindrance between the matrix and the ligand
The high affinity of Cibacron Blue for serum albumin is used routinely in many laboratories for the one-step removal of this major protein from immune sera during isolation of immunoglobulins. The low cost and ready availability of this and other triazine dyes are valuable assets in the large-scale purification of certain enzymes. Affinity chromatography also can be combined with high-performance liquid chromatography (HPLC) by attaching specific ligands to the solid supports used in HPLC. Perhaps overall, the most useful applications are with "general" ligands, such as nucleotides and cofactors. The versatility of one such application was shown recently by Christopher R. Lowe and coworkers at 22
August 26, 1985 C&EN
Cambridge University, England. They used a number of reactive triazine dyes attached to microparticulate silica to separate by HPLC a variety of enzymes, including lactate dehydrogenase, hexokinase, alkaline phosphatase, carboxypeptidase, and L-tryptophenyltRNA synthetase. They demonstrated quite convincingly the exceptional speed, resolution, ease of operation, and selectivity of the technique. Hydrophobic affinity chromatography, as the name implies, exploits the hydrophobic interactions between a protein and an immobilized, hydrophobic ligand. The most commonly used hydrophobic ligands include phenylalkyl moieties and alkyl chains of various lengths. Hydrophobic binding sites have been recognized in certain enzymes and other proteins, such as human pituitary thyrotropin and detergent-solubilized erythrocyte membrane proteins. The deliberate use of such hydrophobic interactions, which ordinarily are considered a source of interference in affinity chromatography, was first exploited, simultaneously and independently, in 1972 by Shmuel Shaltiel of Weizmann Institute and by the late Barend H. J. Hofstee of the Medical Research Foundation in Palo Alto, Calif., for purifying a variety of proteins. Penicillin and other /3-lactam antibiotics kill certain strains of bacteria by covalently attaching themselves to cell-wall proteins, thus preventing cell-wall biosynthesis. Peter M. Blumberg and Jack L. Strominger at Harvard University exploited this observation in 1972 to isolate several bacterial cell-wall proteins by what they called covalent affinity chromatography. They used igarose-bound 6-aminopenicillanic acid as the affinity matrix. The penicillin-binding proteins are covalently attached to the immobilized ligand and can be eluted with a mild nucleophilic agent, such as hydroxylamine hydrochloride. One of the most innovative modifications of affinity chromatography was described recently by Pierre Douzou of the Institut National de la Santé et de la Recherche Médicale in Paris. His method is based on the enzyme-catalyzed reactions' ability to be essentially "frozen" at subzero temperatures while still retaining viable enzyme-substrate complexes. At these low temperatures, immobilized substrates, rather than inhibitors, can be used. Chilled solutions containing high concentrations of salt, which do not affect the enzyme-substrate affinity, cool the samples to temperatures as low as —14°C, thus freezing or arresting the catalysis of the substrate. Later, the adsorbed enzyme can be eluted simply by raising the temperature.
Recombinant DNA applications Many recent applications of molecular biology and recombinant DNA technology require highly purified and biologically active messenger ribonucleic acid (mRNA). In the early 1970s, a number of mRNAs of animal or viral origin were found to contain a region (with up to 150 bases) of the polynucleotide polyadenylic acid (poly A). Immediately after this discovery, a very useful series of affinity chromatographic systems based on immobilized complementary polynucleotides was developed. These systems generally use cellulose to which
an oligomeric complementary nucleotide, such as oligo uridylic acid or oligo deoxythymidylic acid (dT), has been attached. Although such methods produce mixtures of mRNAs, they effectively enrich the mRNA. In 1977, T. Gordon Wood and Jerry B. Lingrel of the University of Cincinnati devised a method for obtaining highly homogeneous and biologically active globin mRNA from mouse erythroid cells. Their method is based on the ability of each specific mRNA to hybridize with a specific complementary DNA (cDNA). It involves the direct cell-free synthesis of the specific cDNA on cellulose to which oligo (dT) has been covalently attached as a primer. Hybridization of unfractionated RNA from mouse erythroid cells with the immobilized cDNA can be used to obtain, in a single step, globin mRNA that retains its biological activity when tested in a wheat germ cell-free lysate system. In 1983, Deepak Bastia and coworkers at Duke University Medical Center combined the techniques of gene fusion and affinity chromatography to isolate a biologically active protein that initiates the DNA replication
Covalent affinity chromatography can be used to purify proteins COOH
CH3 (
KAVWVVWWVCO-NH^"
V / Spacer arm Agarose Adsorption
6-Aminopenicillanic acid Detergent-solubilized crude extracts from the bacterium Bacillus subtills COOH
Protein— serine — O^^/y C'' HN
f
CH3 CH a
\MWWWW co-Nhr Hydroxyiamine hydrochloride Elution Penicillin-binding proteins COOH H O
(
- / H N
CH3 CHft
WA/WWWVCO-NH
D-Alanine carboxypeptidase or other penicillin-binding proteins from crude extracts of the bacterium Bacillus subtilis are bound selectively and covalently to an affinity matrix (agarose-bound 6-aminopenicillanic acid) through the hydroxyl group of a serine residue at the active site of the enzyme. The bound proteins may be eluted from the affinity matrix by cleavage of the ester bond, using a mild nucleophilic agent, such as hydroxyiamine hydrochloride
of an Escherichia coli plasmid. They isolated fused genes for /3-galactosidase and the initiator protein on an affinity column to which an inhibitor of /3-galactosidase, p-aminophenyl-/3-D-thiogalactoside, was covalently attached. Recently, Pennina R. Langer, Alex A. Waldrop, and David C. Ward of Yale University described a simple, general procedure for preparing biotin-substituted nucleic acids that may have a significant impact in many aspects of biomedical and recombinant DNA research. They prepare analogs of nucleotidetriphosphate that contain a biotin molecule covalently bound to the C-5 position of the pyrimidine ring (with a spacer arm). Since these analogs serve as efficient substrates for a variety of DNA and RNA polymerases in vitro, many biotin-containing polynucleotides can be obtained. When such polynucleotides contain low levels of biotin (50 or fewer molecules per kilobase), they exhibit the same denaturation, reassociation, and hybridization properties as the underivatized controls. Both doubleand single-stranded biotinyl polynucleotides are retained selectively and quantitatively on avidinagarose. These approaches should enable great strides to be made in isolating specific DNA and RNA sequences. In addition, these affinity systems may be useful for detecting and localizing specific sequences of polynucleotides in chromosomes, cells, and tissue sections, as well as for gene mapping, and developing selective immunoprecipitation procedures. They add significantly to the range of techniques available for enriching or deleting specific gene sequences in complex mixtures.
Hormone receptors A receptor, which resides on a cell surface or within a cell, is a molecule that recognizes a particular chemical messenger (such as a hormone or a drug), binds to it, and subsequently initiates a series of biochemical events resulting in a characteristic physiological response. The interactions between hormones and receptors are among the strongest and most specific ones known in biological systems. Some of the body's most vital actions and responses, such as blood pressure, muscle movements, and sugar metabolism, are regulated by receptor molecules. The isolation and characterization of hormone receptors are crucial to a clear understanding of the mechanisms of hormonal action. Only after the advent of affinity chromatography did such isolation become experimentally feasible. Because hormone receptors are present in mammalian cells only in vanishingly small quantities (less than 10 ppm on a dry-weight basis) and because they are relatively unstable, traditional methods for isolating and purifying proteins have been of only limited value, especially for those water-insoluble receptors that are normally membrane-bound and thus require the continued presence of detergents to maintain them in solution. Even with various technologial advances, such as affinity chromatography, only a few cell receptors have been purified successfully. The insulin receptor was the first mammalian hormone receptor to be purified. This advance was made in August 26, 1985 C&EN
23
Special Report 1973 by Cuatrecasas, then at Johns Hopkins University, who used affinity chromatography on insulin-agarose derivatives. More than a 500,000-fold purification is necessary to achieve a homogeneous preparation of the insulin re ceptor from liver cells. The formidable nature of this process can be contrasted with the purification of an other membrane-bound receptor, the acetylcholine re ceptor of the electric organ of the eel, which requires only a 50- to 1000-fold purification (depending on the source) to attain homogeneity. The experience gained from purifying the insulin receptor has been used widely to study other membrane-localized hormone receptors that require detergents for solubilization. Various hormones, such as insulin, epinephrine (adrenaline), and nerve-growth factor, retain some of their biological properties when exposed to cells in a form that cannot enter the cells, such as when attached covalently to insoluble polymer or beads or to large soluble polymers. The action of epinephrine is mediated by both α and β types of adrenergic receptors. In 1974, J. Craig Venter and coworkers at the University of California, San Diego, used an epinephrine analog coupled to glass beads to produce ^-adrenergic responses (such as changes in the heart's beating rate) in heart cells. More recently, a sol uble copolypeptide containing covalently attached epinephrine was shown to activate the a-adrenergicmediated stimulation of phosphorylase, Ca 2+ fluxes, and gluconeogenesis in isolated liver cells. The polymeric epinephrine also could induce biological effects in iso lated perfused rat livers. Thus, it appears that at least some receptor-mediated responses can be produced without the hormone's penetrating the cell. Recently, Thomas C. J. Gribnau at Catholic University, Nijmegen, the Netherlands, confirmed Cuatrecasas' original finding in 1969 that agarose derivatives of in sulin (when properly prepared and washed) can stim ulate certain insulinlike responses (such as glucose transport) in isolated fat cells under conditions where
immobilized concanavalin A mitogenically activates B-lymphocytes Concanavalin A
+
Θ— Θ
B-Lymphocyte
No activation
Activated B-Lymphocyte Concanavalin A
Immobilization of concanavalin A (a mitogen) and other lectins on agarose beads or other supports confers mitogenic activity by allowing microaggregation of lectin receptors on the B-lymphocyte cell surface. Free concanavalin A, although capable of binding to the cell surface receptors, does not produce receptor aggregation and mitogenic activation of B-lymphocytes
the leakage of insulin cannot account for the observed effects. Since only the insulin present on the surface of the agarose bead is potentially able to interact with cell-surface recepturs, and since relatively few beads (50 to 100 per incubation flask) are required, the agarosederivatized insulin must have extraordinarily high ac tivity. The mechanism by which immobilized insulin interacts with receptors and triggers responses is likely to be different from that for the native hormone. Strong evidence exists for the lateral mobility of cell-surface hormone receptors and for the role of receptor microaggregation in triggering at least Affinity chromatography can be used to purify various the immediate, fast responses of hormones. In all likelihood, the membrane-bound receptors potency of the agarose derivatives Receptor Source Ligand Solubillzlng agent of insulin (and other hormones) Acetylcholine Electric eel Non ionic detergents, Cobratoxin stems from their polyvalence. Pos deoxycholate sibly, high-affinity local patches Epidermal growth Placenta Plant lectins Nonidet P-40 factor (clusters) of receptors are formed at Digitonin Alprenolol Epinephrine Erythrocytes the points of contact, and only a few (adrenaline) of these may be needed per cell to Growth hormone Triton X-100 Growth hormone Liver generate maximal responses. If the Human chorionic Triton X-100 Human chorionic Testes receptor clusters do not disaggre gonadotrophin gonadotrophin gate rapidly after detachment from Immunoglobulin Ε Nonidet P-40 Immunoglobulin Ε Basophilic cells the bead, the effect could persist for Insulin, concanavalin A, Liver, fat cells, Triton X-100 Insulin some time, even if the receptors are or wheat germ placenta agglutinin not occupied with hormone. Thus, Growth hormone Prolactin Mammary gland Triton X-100 the beads could be acting almost Thyrotropin-stimulating ThyrotropinThyroid gland Lithium catalytically in triggering receptor hormone stimulating diiodosalicylate responses. hormone One dramatic example of this is
24
August 26, 1985 C&EN
the finding that immobilized concanavalin A, being polyvalent, activates the mitogenesis of B-lymphocytes, whereas the free compound does not. Such findings are consistent with recently evolving evidence that the hormonally induced uptake of surface receptors is not likely to be required for the mediation of the rapid, immediate actions of hormones. On the other hand, it has been suggested that such uptake of receptors (but not the hormone) by the cell may be necessary for the more-delayed hormone actions, such as mitogenesis and cell proliferation. Studies of immobilized hormones can help illuminate various basic processes in cell biology. Furthermore, such derivatives, especially those of hormones with soluble polymers, may find use in treating diseases be cause of their altered potency, improved distribution in the body, and the more-effective processes by which they ultimately are degraded and removed from the body.
Steroid hormones Estrogens and progesterone, the female sex hormones synthesized in the ovary, play a vital role in reproduc tion and other aspects of female physiology. Estrogens, like various other steroid hormones, act through specific macromolecular receptors present in the extranuclear space of target-tissue cells. In contrast to the mem brane-bound receptors for peptide hormones, these re ceptors are soluble in normal physiological buffers. Es trogen receptors are most abundant in the uterus, amounting to as much as 10 ppm in intact tissue. Because of the scarcity of the estrogen receptor in bi-
Some water-soluble cytoplasmic receptors can be purified by affinity techniques Receptor
Cyclic AMP Estrogen Glucocorticoid Progesterone Vitamin D
Source
Skeletal muscle Calf uterus Rat liver Chick oviduct Chick kidney
Ligand
Cyclic AMP Estradiol, diethylstilbestrol Dexamethasone Progesterone, DNA Hydroxycholecalciferol
Lectin-agarose affinity columns are used to purify several glycoproteins Lectin
Concanavalin A
Ricinus communis Wheat germ agglutinin
Glycoproteins
α-Antitrypsin, herpes-specific membrane glycoproteins, horseradish peroxidase, human alkaline phosphatase, interferon, immunoglobulin A, receptors for insulin and epidermal growth factor, porcine enteropeptidase, rat brain glycopeptides, thyrotropin α-Fetoprotein Erythropoietin, glycophorin A, receptors for insulin and somatomedin C, retinal glycoproteins
ological materials, together with its very high affinity for estradiol and its relative instability in solution, its purification by affinity chromatography has been very appealing. A number of difficult problems, however, have been encountered in this research. For example, the receptor cannot be eluted from an estrogen-affinity column in an active form by simply modifying the eluting buffer's properties (such as pH, ionic strength, and the presence of protein dénaturants). Such methods are either not effective, or they cause irreversible denaturation of the receptor. The receptor can be eluted from such columns, however, by using buffers that contain estrogens. It can be purified by affinity chromatography on agarose derivatives substituted through the carboxyl group of 17acarboxymethyl-17/3-estradiol. Agarose derivatives substituted through the A ring of the steroid molecule are ineffective in binding the receptor, indicating that the A ring is the business end of the molecule. Recognizing that the native estrogen receptor binds to heparin reversibly, Giovanni A. Puca of the University of Naples, Italy, has used immobilized heparin to partially purify this receptor. In current practice, estrogen receptors are purified by a two-stage affinity chromatographic method, first on heparin-agarose, followed by the use of an estradiol-agarose column.
Plasma-membrane proteins Lectins, also known as plant agglutinins, are a family of cell-agglutinating proteins that are ubiquitous in plants and bind to specific sugar moieties. Among the best-known lectins are concanavalin A (specific for α-mannose and α-glucose), wheat germ agglutinin (specific for N-acetylglucosamine), and peanut agglu tinin (specific for galactose). Lectins are used widely in affinity chromatography because they bind strongly and selectively to complex glycoproteins. In addition, com plete elution is easy with solutions of simple sugars (thus adding to the selectivity of the overall procedure). In contrast to the proteins present in cell cytoplasm, many of the proteins associated with cytoplasmic membranes and exposed to the cell exterior are glyco proteins. Hence, appropriately selected plant lectins are being used increasingly to study and separate solubilized cell-membrane components by affinity chromatography. This approach was first used in 1973 by Cuatrecasas to purify detergent-solubilized, membrane-bound insulin receptors using agarose containing wheat germ agglu tinin or concanavalin A. Recently, Morley D. Hollenberg and associates of the University of Calgary, Canada, developed an extension of this approach. Their method is especially valuable for identifying and purifying receptors that are sensitive to detergents. They showed that detergent-solubilized receptors for epidermal growth factor and transcobalamin II, which cannot be shown to bind their ligands in the presence of detergents, nevertheless are adsorbed well (and specifically) on lectin-agarose beads. Once the detergent is removed after adsorption, the binding ac tivity is restored, and detection of the receptor becomes quite simple. This approach, which should be generally applicable, permits study of the chromatographic beAugust 26, 1985 C&EN 25
Special Report
In scanning electron micrograph, human red blood cells are bound to wheat germ agglutinin-derivatized agarose beads. This agglutinin has high affinity for N-acetylglucosamine components of the surface glycoproteins of red blood cells havior of detergent-solubilized glycoprotein receptors and probably will facilitate efforts to purify them. Based on differences in the glycoprotein composition of cell-surface proteins, immobilized lectins can be used to separate intact cells and to purify plasma-membrane vesicles from other subcellular organelles. J. Kambayashi and colleagues at Osaka University, Japan, have purified plasma-membrane vesicles of human platelets using agarose-immobilized wheat germ agglutinin. One of the most elegant uses of immobilized lectin is in separating inside-out and right-side-out vesicles of erythrocyte (red blood cell) membranes. Conversely, of course, lectins themselves can be purified on affinity adsorbents containing lectin-specific simple sugars or glycoproteins. Some of the polysaccharide supports used in gel-permeation chromatography, such as Sephadex or agarose, can be used as natural adsorbents for selectively purifying certain lectins. A cell characteristically has an external coat, called the plasma membrane, containing a number of exposed proteins that serve a variety of biological functions. These functions include cell-cell recognition and the receiving and transmitting of hormonal signals. It is necessary sometimes to isolate and study these proteins by methods not based on their functional role. One general method for labeling and isolating the external surface proteins of cells has been described re26
August 26, 1985 C&EN
cently by Avner Rotman and Shoshana Linder of Weizmann Institute, who have been isolating the plasma-membrane proteins of blood platelets. In their procedure, dinitrophenyl-/3-alanine hydrazide reacts with and labels the periodate-oxidized sugar and sialic acid residues of the externally exposed glycoproteins. After the platelets are solubilized with a nonionic detergent, the labeled glycoproteins are isolated on affinity columns packed with agarose-bound antidinitrophenyl antibodies. The isolation and separation of plasmamembrane glycoproteins on agarose-immobilized plant lectins provide an attractive alternative approach. The term affinity electrophoresis, first proposed by Thorkild C. Bog-Hansen of the University of Copenhagen, Denmark, refers to the electrophoretic separation of proteins in the presence of a specific ligand. Although the method is a logical extension of the previously developed counterimmunoelectrophoresis and rocketimmunoelectrophoresis procedures, it provides a valuable means of separating and identifying proteins. Affinity electrophoresis is especially valuable as a simple method for identifying a specific protein in a complex mixture from biological fluids or tissue extracts. In several laboratories, attempts are under way to explore the method's potential use for analyzing isoenzymes, protein antigens, and antibodies from patients' serum samples or proteins in urine and saliva. Affinity electrophoresis often is used to explore the feasibility of using affinity chromatography to purify a certain protein or to determine quantitatively the affinity between a protein and its specific ligand. Since about 1960, polyacrylamide-gel electrophoresis has been used routinely for separating and determining the molecular-weight distribution of protein or DNA molecules in a mixture. The protein bands separated on the gel usually are made visible by a color stain. However, identification of a protein on the gel is possible only if a convenient biochemical method for the specific tagging of the protein is available. The identification can be simplified if the specific protein is transferred selectively to a filter paper that has been derivatized with a specific ligand. Stanley N. Cohen and coworkers at Stanford University use filter papers chemically derivatized with a specific antibody, antigen, or lectin. Proteins that interact with the covalently coupled affinity ligand are transferred specifically to the filter paper when it is overlaid on a gel containing the separated proteins. The transferred proteins, if radiolabeled, are detected by autoradiography. An unlabeled protein can be detected by a radiolabeled specific antibody or, alternatively, by treatment with the antibody, followed by treatment with radiolabeled Staphylococcus aureus protein A. George Stark and coworkers, also at Stanford, have developed a similar blotting technique with diazotized filter paper that transfers all of the proteins from an electrophoretic gel. The transferred proteins then can be identified with specific radiolabeled ligands or with an antibody in combination with radiolabeled protein A. Some functions of membrane-associated receptors are best studied in their natural membrane environment.
The two types of vesicles from red blood cell ghosts can be separated by immobilized lectins Lectln
Glycoproteins Agarose
®*1 Red Wood cell ghost
Lectin
Osmotic
+
shock
© ©> Membrane vesicles Red blood cells from which the pigment has been removed are commonly called ghosts. Brief treatment of red cell ghosts in buffers of very low ionic strength (osmotic shock) causes their membranes to rupture. The fragmented membranes fuse again to make two types of small vesicles. One group of vesicles with the original glycoproteins on the out-
Ion-translocation phenomena and the influence of local anesthetics on the cholinergic receptor, for example, cannot be studied with the solubilized or purified receptor. Furthermore, solubilization of this membranebound receptor with nonionic detergents alters agonist binding to a certain extent and also partially dissociates one of the subunits of the receptor. Classical centrifugation methods permit partial purification based on the size and density of membrane fragments, but they are ineffective for fractionation on the basis of binding-site composition. Recently, Steven D. Flanagan and associates at the City of Hope Research Institute in Duarte, Calif., purified membrane fragments rich in cholinergic receptor by a novel affinity-partitioning technique. The source of their receptor was the electric organ of various electric fish, such as Torpedo californica. These researchers purified receptor-containing membrane fragments by the differential distribution of these fragments between two aqueous phases, each enriched with a different highly water-soluble polymer. To induce such separations, a ligand known to interact specifically with the receptor was covalently attached to one of the two polymers. A bis-quaternary ammonium ligand covalently coupled to poly(ethylene glycol) allows membranes containing cholinergic receptors to be enriched in one of the phases. Such affinity-induced partition methods, combined with multiple extraction procedures, such as countercurrent distribution, provide an excellent approach for purifying specific membrane fragments. The method probably will be useful in separating and purifying other subcellular components and even for fractionating intact cells. Werner Muller and coworkers at the University of Bielefeld, West Germany, have studied the use of various organic dyes to bind selectively to specific base pairs and
- *
Lectin
•U ©
©
Lectin- specific sugar
e Inside-out vesicles
«
&
Right-side-out vesicles
side surface (right side out) can bind to plant lectins, whereas the inside-out vesicles, because of lack of external glycoproteins, cannot. The lectin-bound vesicles are selectively eluted with a solution of lectin-specific sugar. This method is useful in studying the biochemical characteristics of the inside and outside surfaces of eukaryotic cells
thus separate high-molecular-weight DNA chains. Covalent attachment of one of these dyes to poly(ethylene glycol) allows DNA to be selectively partitioned into one of the two phases of a PEG-dextran system. Certain triphenylmethane dyes show strong preference for DNA having two adjacent adenine-thymine pairs in alternating arrangements, whereas others exhibit high specificity for DNA with adjacent guaninecytosine pairs. Some of these dyes can even discriminate between closed-circular and linear-open-chain DNA molecules. These base- and sequence-specific dyes also have been used to study DNA by affinity chromatography and affinity electrophoresis.
Cell separations Unlike protein fractionation, methods available for separating and isolating cells of a restricted class or function are very limited. The relative fragility and limited viability of isolated cells further restrict manipulations by currently available fractionation procedures. Moreover, criteria for the purity of cells are difficult to define. Even when isolated cells appear identical under the microscope, they actually may have different structural and functional properties. Much progress has been made in methods for separating cells, however, particularly free-floating cells such as those in blood (for example, leukocytes and lymphocytes) or those obtained by tissue-culture methods. These procedures are not especially useful, though, for separating cells unless the uniqueness of the cells can be identified and highlighted by labeling some component (such as a surface antigen or receptor) with a fluorescent or chromophoric tag. Affinity chromatography also can be of value for separations based on unique surface markers, including hormone receptors, specific glycoproteins, or surface immunological markers. In 1969, Hans Wigzell at the Karolinska Institute in Stockholm was one of the first to August 26, 1985 C&EN
27
Special Report attempt the isolation of antibody-forming immune cells on an antigen-coated affinity adsorbent. He adsorbed the antibody-forming cells dispersed from the lymph nodes of a mouse immunized with human serum albumin on glass beads coated with the albumin. The adsorbed cells were eluted by gently shaking the beads, thus causing a substantial enrichment of cells capable of producing antialbumin antibodies. Shortly after the publication of Wigzell's report, Leon Wofsy of the University of California, Berkeley, de scribed a similar affinity method using large derivatized polyacrylamide beads to purify cells at various stages of a particular immune response. More recently, Gerald M. Edelman and associates at Rockefeller University ex tended this procedure by separating cells according to their ability to bind specifically to tightly strung nylon fibers derivatized with such substances as antigens, antibodies, or lectins. The cells are removed by plucking the fibers or by using buffers containing a specific ligand or lectin-specific sugars. This strung-fiber method per mits direct observation of the process under the micro scope, as well as better control of various experimental manipulations. The technique also may be of value for concentrating dilute suspension of viruses and various parasites. Saul Roseman and coworkers at Johns Hopkins have been studying a number of new methods for separating cells. The Hopkins group has shown that, in many cases, cell-cell recognition and adhesion are mediated by one or more specific carbohydrates present on the cell sur face. They find that isolated liver cells from different species recognize and bind to specific and sometimes different sugar moieties. Thus, rat liver cells bind to beads containing immobilized galactosides, whereas chicken liver cells bind specifically to immobilized Nacetylglucosamine. Along this line, Cuatrecasas and Susan W. Craig, while at Johns Hopkins, found that cholera toxin binds to its cellular receptor (the ganglioside GM1) multivalently. They discovered this by observing that agarose beads derivatized with GM1 can very effectively and effi ciently adsorb lymphocytes, provided cholera toxin is attached to their surface. Diane J. Dvorak and colleagues at the University of Queensland in Brisbane, Australia, recently demonstrated the feasibility of affinity cell purification by adsorbing the dispersed sympathetic ganglion neurons of chick embryos on agarose beads containing α-bungarotoxin, a neurotoxin specific to the acetylcholine receptor. The selectively adsorbed cells are removed from the beads by mild digestion with trypsin. Although many separations of mammalian cells by affinity chromatography have been reported, it is sur prising that the separation of bacterial cells by this method has been largely overlooked. Recently, Thomas Ferenci and coworkers at the University of Sydney, Australia, used affinity chromatography to enrich or isolate rare genetic mutants of bacteria in which various surface components were altered. Recent rapid innovations in monoclonal antibody and hybridoma technology probably will lead to dramatic advances in cell separations. Since it is now possible to 28
August 26, 1985 C&EN
produce highly selective antibodies directed to unique cell-surface determinants, future separations are likely to involve much more selective, homogeneous, and high-affinity probes for pulling out of complex sus pensions even minor subtypes of specialized normal or malignant cells. Separation of cell organelles by density-gradient centrifugation is more effective if the density of the desired organelle is increased by selective association with higher-density particles. A method known as af finity-density perturbation, first proposed in 1972 by Donald F. G. Wallach and coworkers at Tufts-New En gland Medical Center, Boston, uses bacteriophage covalently derivatized with concanavalin A to introduce density alterations of glycoprotein-bearing membrane fragments. The phage-associated membranes, being heavier than either of the components, then are sepa rated easily by centrifugation in a density gradient containing cesium chloride. The specifically adsorbed membranes are separated from phage simply by disso ciating them with a lectin-specific sugar, followed by differential centrifugation. The method is not limited to the use of concanavalin A or of bacteriophage. Any specific ligand, such as a hormone or a membrane protein-specific antibody, may be used instead of concanavalin A. Furthermore, other materials, such as latex beads, may be used in place of bacteriophage. Although this technique has not been widely exploited, its principles are suitable for separat ing intact cells as well. Howard M. Katzen and colleagues at Merck Institute for Therapeutic Research in Rahway, N.J., have used immobilized insulin-agarose to demonstrate the pres ence of insulin receptors on the surface of fat cells. De pending on the quantity of insulin-agarose added and the degree of substitution of insulin on the beads, the fat cell-agarose complex spontaneously settles at the bottom of a test tube or remains floating (with agarose beads attached) in a physiological buffer.
Viruses and phages The application of affinity chromatography to the purification of viruses and phages, unlike that of cells and cell membranes, is still in its infancy. Only a few viruses have been isolated by this technique. Never theless, the relative scarcity of virus particles in an in fected tissue, along with the difficulty of growing vi ruses in large amounts in tissue culture, makes them ideal candidates for isolation by affinity chromatogra phy. Some major limitations, however, include lack of knowledge of unique biochemical interactions and li gand specificity, together with difficulties in producing specific antibodies to viral particles. Aleutian mink disease, influenza, and hepatitis Β vi ruses are perhaps the best studied examples. In 1971, Cuatrecasas and Gennaro Illiano, then both at Johns Hopkins, found that influenza virus could be adsorbed specifically to an oxamic acid-agarose column, which acts as an affinity adsorbent for the viral-surface enzyme neuraminidase. In 1973, Alan J. Kenyon, then at the University of Minnesota medical school in Minneapolis, isolated
Affinity column can remove unwanted substances from the blood Column containing an affinity ^adsorbent, such as agarose, to which human serum albumin has been attached Inflow sampling site Li-
t i 1=1
Arterial line
Outflow sampling site
•M
Venous line Peristaltic pump This extracorporeal hemoperfusion circuit can remove un desirable substances in the blood, such as poisons, over dosed drugs, or excessive amounts of natural substances, by passage of the blood through an affinity column. Human serum albumin, attached to agarose beads, binds tightly to the natural substance bilirubin and has been tested in labo ratory animals for treating jaundice
Aleutian mink disease virus on affinity adsorbents to which viral antibodies had been attached covalently. The specific antibody used for this purpose was obtained from the serum of chronically infected mink. Similar studies of hepatitis Β virus have shed considerable light on the possible antigenic determinants of these viral bodies, although the isolation of viral particles in active infective form thus far has not been possible. Similarly, foot-and-mouth-disease, Semliki-forest, and tobacco-mosaic viruses have been isolated by affinity chromatography with the use of immobilized specific antibodies. In the future, the development of monoclo nal antibodies to viral antigens may overcome the dif ficulties associated with past research and provide highly specific antibodies for identifying and isolating various viruses and their subclasses. Among the bacteriophages, only T4 phage appears to have been purified by affinity chromatography. In 1976, Elzbieta Romanowska and coworkers at the Polish Academy of Sciences in Breslau used a biospecific in teraction between certain lipopolysaccharides and bac teriophage for such a purification.
Affinity therapeutics Of considerable potential value are bioselective af finity resins for selectively removing harmful or path ological substances from the blood or intestine. Until recently, however, activity in this area has been rela tively slight. The potential medical applications of these concepts are self-evident. In fact, in 1971 and again in 1972, when both of us were at Johns Hopkins, we applied for federal
grants for in-vitro feasibility studies of what we referred to as "affinity therapeutics/' We suggested the possible removal of iron in overdose patients by using selective resins (such as desferroxamine-agarose) that might be taken orally. We also suggested the use of immobilized antibodies to remove (by extracorporeal systems) digoxin or other drugs or the poisons most commonly used in suicide attempts. Moreover, we proposed studying the feasibility of developing materials to selectively adsorb cholesterol from the gastrointestinal tract and to remove circulatory anti-DNA antibodies in patients with sys temic lupus erythematosus. Although our funding re quests were modest, since we were proposing only bio chemical and pharmacological feasibility studies, we were turned down, presumably because these objectives were too speculative at the time. Since then, various attempts have been made to re move materials from the blood of animals through ex tracorporeal shunts containing bioselective adsorbents. Systems containing activated charcoal have been used for some time to remove various toxic substances nonspecifically from patients' blood, although most of these efforts have been plagued by the simultaneous removal of desirable materials or, more seriously, by the de struction of platelets. Perhaps platelet destruction now can be prevented by using the potent antiplatelet-aggregating agent prostacyclin. Davis S. Terman and colleagues at Baylor College of Medicine have used extracorporeal shunts containing immobilized DNA to remove circulating DNA anti bodies in immunized rabbits. They also have removed antialbumin antibodies nearly quantitatively (with very few side effects) from immunized dogs by using micro capsules to which albumin has been attached with glutaraldehyde. Isaac Schenkein and coworkers at New York University have achieved similar results in rabbits, using columns containing albumin attached to agarose or cellulose. To demonstrate the feasibility of removing the en dogenous poisons associated with liver failure, Bruce F. Scharschmidt and colleagues performed some important experiments at the National Institutes of Health on rats that were jaundiced by the congenital absence of the enzyme glucuronyl transferase or by obstruction of the biliary duct. Their extracorporeal columns contained human serum albumin (which binds bilirubin very tightly) attached to agarose. The results were rather dramatic; more than 50% of the radioactive bilirubin was removed within one hour in the enzyme-deficient rats and more than 95% was removed in rats whose biliary ducts had been obstructed surgically. These studies should encourage further exploration of affinity therapeutics. Certainly, a variety of diseases can be treated by such approaches. With some imagi nation, one can visualize applications such as the re moval of circulating endotoxins (in infections), auto antibodies (in arthritis and other autoimmune diseases), lipids (in hyperlipemic states), intact cells (as in leukemias or bacteremias), or particles (such as viruses). Fur thermore, orally effective and innocuous adsorbents might remove bacterial toxins (as in cholera). Other medical applications could result, at least in August 26, 1985 C&EN
29
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Special Report part, from these techniques. For example, they might lead to the production of polymeric hormones or drugs that have increased potency, greater tissue selectivity, or longer duration of action. Several labs are studying addition products of hormones or antibodies with bac terial toxins, cytotoxic drugs, or destructive radioisotopes as selective targeting agents to combat tumors. The boundaries of affinity-adsorbent methods merge with other closely related fields in what may be broadly termed solid-phase biotechnology. For some time now, insoluble matrices have been standard tools in peptide synthesis (the Merrifield technique) and in peptide se quencing (the Laursen technique). In addition, the al ready wide use of immobilized enzymes in research on the basic mechanisms of enzyme function and cataly sis—as well as their use in enzyme reactors for the in dustrial production of chemicals—likely will expand further as improvements in polymer chemistry and in the production of the enzymes themselves (through DNA recombinant technologies) open up new frontiers. Solid-phase enzyme immunoassays are becoming stan dard procedures in clinical labs, and scientists can expect even more innovations leading to greater selectivity, ease of use, and analytical sensitivity. D
Selected readings BOOKS Chaiken, I., Wilchek, M., Parikh, I., Ed., "Affinity Chromatography and Biological Recognition," Academic Press, New York, 1984. Jacoby, W., Wilchek, M., Ed., "Methods in Enzymology," Vol. 34, Academic Press, New York, 1974. Lowe, C. R., Dean, P. D. G., "Affinity Chromatography," WileyInterscience, London, 1974.
Schott, H., "Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins," Marcel Dekker Inc., New York, 1985. Scouten, W. H., "Affinity Chromatography," John Wiley & Sons, New York, 1981. Turkova, J., "Affinity Chromatography," Elsevier, Amsterdam, 1978. ARTICLES AND REVIEWS Cuatrecasas, P., "Affinity chromatography of macromolecules," in "Advances in Enzymology," Vol. 36, Meister, Α., Ed., John Wiley & Sons, New York, 1972. Cuatrecasas, P., "Protein purification by affinity chromatography; Derivatizations of agarose and polyacrylamide beads," J. Biol. Chem., 245, 3059(1970). Cuatrecasas, P., Anfinsen, C. B., "Affinity chromatography," Ann. Rev. Biochem., 40, 259 (1971). Cuatrecasas, P., Hollenberg, M. D., "Membrane receptors and hormone action," Adv. Protein Chem., 30, 251 (1976). Parikh, I., Cuatrecasas, P., "Affinity chromatography in immu nology," in "Immunochemistry of Proteins," Vol. 2, Atassi, M. Z., Ed., Plenum Press, New York, 1977. Sharma, S. K., Mahendroo, P. P., "Affinity chromatography of cells and cell membranes," J. Chromatogr., 184, 471 (1980). Venter, J. C, "Immobilized and insolubilized drugs, hormones, and neurotransmitters—Properties, mechanisms of action, and applications," Pharmacol. Rev., 34, 153 (1982).
Reprints of this C&EN special report will be available at $5.00 per copy. For 10 or more copies, $3.00 per copy. Send requests to: Distribution, Room 210, American Chemical Society, 1155— 16th St., N.W., Washington, D.C. 20036. On orders of $20 or less, please send check or money order with request.
kins school of medicine from 1970 to 1975. He has received several scientific awards, including the John Jacob Abel Award in Pharmacology in 1972, the Eli Lilly Award of the American Diabetes Association in 1975, and the Goodman Gilman Award in 1982 for his achievements in hormone-receptor research. He was elected to the National Academy of Sciences in 1982 and NAS's Institute of Medicine in 1981. In addition to his interest in the mechanism of insulin action, he has made important contributions to the study of the structure and mechanism of action of other peptide hormones and drug receptors. Indu Parikh {right) is associate head of the department of molecular biology at Wellcome Research Laboratories. Born in India, he received a Ph.D. degree in 1966 from the University of Zurich, Switzerland. Later, he did postdoctoral research at Weizmann Institute of Science in Rehovot, Israel, and at the National Institutes of Health. In 1970, he joined Johns Pedro Cuatrecasas (left) is vice president of the research, de Hopkins school of medicine as assistant professor in both the velopment, and medical division and head of the department department of pharmacology and experimental therapeutics of molecular biology at Wellcome Research Laboratories, Re and the department of medicine. He serves on the editorial search Triangle Park, N.C. Born in Spain, he obtained his M.D. boards of several biochemical journals and on the board of di in 1962 from Washington University school of medicine and rectors of the Alopecia Areata Research Foundation. He has served an internship and residency at Johns Hopkins Hospital. been at Wellcome Research Laboratories since 1975, where After gaining extensive experience in endocrinology, protein he is studying the mechanism of action of estrogens and de chemistry, and enzymology at the National Institutes of veloping new technologies for targeted drug-delivery sys Health, he taught pharmacology and medicine at Johns Hop- tems. 32
August 26, 1985 C&EN
