Ind. Eng. Chem. Prod, Res. Dev. 1904, 23, 17-21
Agar and Agarose: Indispensable Partners in Biotechnologyt Donald W. Renn FMC Corporation, Rockland, Malne 0484 1
The algal polysaccharides, agar, agarose, carrageenan, and algin, have played and are continuing to play a significant role in advancing the state of the art in biotechnology. Agar and its more neutral component, agarose, have been and continue to be particularly Important in biomedical research and diagnosis of disease states. More recently, agar has been used extensively as a medium for cloning genetically engineering microorganisms, as well. At present, agarose is the only known thermoreverslble, ion-independent gelling agent. Agarose can be prepared with consistent properties, is essentially neutral chemically, and forms strong aqueous gels at low concentrations. Because of these characteristics, mapping of gene fragments and separation prlor to insertion In plasmids, as well as providing an excellent medium for plant and animal cell culture and affinity separations, have been added to the ever-increasing number of applicatlons for agarose. Low gelling-melting temperature hydroxyethyl, as well as other unique derivatives, have further extended agarose’s indispensibllity in biotechnology.
Introduction Because of their unique properties, the algal polysaccharides, agar and agarose, have played and are continuing to play increasingly important roles in various areas of emerging biotechnology. Before looking at the applications and the properties of agar and agarose which make these possible, a brief look at the major classes of marine algae and hydrocolloids extracted from them (Figure 1) is warranted. The four major classes of seaweeds are the Rhodophyta or red algae, Phaeophyta or brown algae, Cyanophyta or blue-green algae, and Chlorophyta or green algae. Only the red and brown algae are currently sources of commercial products of significant value. Three types of carrageenan, other miscellaneous sulfated galactans, and agar, from which agarose is derived by purification, are obtained from red algae, but not from the same species. Algin, also an important commercial product, is obtained from a number of species of brown algae. Agar Agar, a mixture of galactan derivatives extracted from certain red seaweeds, has achieved commercial importance because of its gel-forming properties. Worldwide production of agar is estimated (J. Commerce, Oct 7, 1982) to be about 11million lb per year, with chief producers being Japan, Spain, Taiwan, Korea, Morocco, Chile, Portugal, and the United States. About two-thirds of this is used in foods, medicines, and as a component of dental impression materials. Most of the remainder is used for bacteriological media. Less than 1% is used as a raw material for agarose production. Biotechnology is defined in Webster’s New World Dictionary as “the use of the data and the techniques of engineering and technology for the study and solution of problems concerning living organisms.” It was 1882 when Dr. Robert Koch formally announced agar as a new solid culture medium for microorganisms following his now-famous experiments on tuberculosis bacteria. Thus, it can be said that agar was first used in a biotechnology application in the 1880s. Amazingly, the most significant use for nonfood and pharmaceutical agar has not changed in 100 years! It is still the medium of choice for general microbiological growth and identification. With the advent ‘Presented as an invited paper at the Symposium on the Industrial Utilization of Polysaccharides, 185th National Meeting of the American Chemical Society, Seattle, March 21, 1983.
of recombinant DNA and cell fusion techniques, much of the selection, cloning, and propagation of modified bacteria and yeasts is being done on agar. Agarose Agar does, however, have its limitations because of varying and ill-defined ionic moieties. The concept that agar is comprised of neutral “agarose” and ionic “agaropectin” is an oversimplification which persists throughout most of the current literature. Most of the components of agar do have the agarobiose backbone (Figure 2) or a precursor. Although not always present concurrently, sulfate ester, methoxyl, ketal pyruvate, and carboxyl groups can appear on the agarobiose backbone in an almost infinite number of combinations. The conditions used for separation determine in which fraction specific molecules appear. Duckworth and Yaphe (1971), as a result of their comprehensive chromatographic and enzymatic studies, recommended as a practical definition of agarose: “,.. that mixture of agar molecules with the lowest charge content and, therefore, the greatest gelling ability, fractionated from a whole complex of molecules called agar, all differing in the extent of masking with charged groups.” Most commercial processes for isolating agarose from agar are based on differences in solubilities and/or chemical reactivity associated with the anionic character of the “agaropectins.” The physical and chemical properties of each agarose preparation reflect the seaweed source, including location and stage of growth cycle, agar recovery procedure, and process used to isolate the agarose. In order to assure lot-to-lot agarose equivalence, and thus permit reproducibility for a given assay, a number of characterization parameters have been explored and methods developed for their quantification. The most important of these are gel strength, gelling and melting temperatures, and electrical properties of the gel. At present, agarose is the only known thermoreversible, ion-independent gelling agent. Although no idealized agarose preparation has yet been reported which contains no anionic substituents, some types of agarose are sufficiently devoid of charged residues to be essentially neutral and to exhibit virtually no nonspecific protein reactivity. Because agarose gels water at 1.0% or less, mechanically stable gels with large pores are easily formed. Figure 3 shows the relationship of concentrations of aqueous gels of one type of agarose with exclusion limits of spherical proteins. 0 1984 American Chemical Society
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Ind. Eng. Chem. Prod. Res. Dev., Vol. 23. No, 1. 1984 GEL TENPERATURE (SOL)
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Figure 1. Hydrocolloids from different classes of marine algae.
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Figure 4. Variation of gelling and melting temperatures with different types of agarose.
Figure 2. Agarabiose: basic repeating unit of agarose,
Figure 5. Electroendosmosis (EEO).
Figure 3. Apparent molecular exclusion limits as a function of agarose gel concentration by use of agarose gel chromatography.
Gelling and melting temperatures of any agarose preparation are not identical, due to a molecular interaction phenomenon called hysteresis. F m e 4 shows melting and gelling temperatures for four different types of agarose. The temperature a t which an agarose solution gels under given conditions has been found hy Guiseley (1970) to he directly related to the methoxyl content, with very few exceptions. Thus, agarose of Gelidium origin having inherently fewer methoql substituents gela within the range of 34-38 "c,whereas that from Gracilaria gels at 4&52 OC. These measurements were obtained under dynamic conditions, cooling at the rates of 0.5 OC/min. Gelation occurs at somewhat higher temperatures when considerably slower cooling rates are used. Electroendosmosis (or EEO) is an important factor where applications involve using agarose gels in an electric field. Although predominantly neutral, the agarose matrix
contains some anionic residues, sulfate, and pyruvate. Associated with these residues are hydrated counterions (Figure 5). When an electric potential is applied across an agarose gel, the counterionsmigrate toward the cathode carrying their water of hydration and any neutral sample molecules with them. Thus, there is a net flow of water in the gel toward the cathode, while the fixed anionic groups in the matrix are unable to move. The liquid flow is termed electroendosmosis. Some applications depend on this property. Biotechnology applications of agarose and ita derivatives fall into five main categories: (1)electrophoresis, (2) immunology, (3) microorganism culture, (4)chromatography, and (5)immobilized enzyme and cell technology-along with various combinations among the five. All reported applications cannot be covered in this brief review, and therefore, only a few will be highlighted. Electrophoresis. Agarose gels containing appropriate buffers provide excellent media for separation of polyelectrolytes, particularly proteins and nucleic acids and their derivatives, by charge and/or mass by use of an electric potential. Separations by charge are based on differential rates of migration of charged particles toward the oppositely charged electrode when an electric potential is applied across the gel. Electrophoretic separation by mass or molecular sue depends on the relative ability of particles to migrate through the pores of the gel matrix. The smaller the molecule, the less restriction, and, therefore, faster movement. This type of electrophoretic sorting is frequently referred to as molecular sieving electrophoresis. Different proteins contain different charge to mass ratios. Sometimes chemicals such as the detergent sodium dodecyl sulfate (SDS) are used to minimize or completely mask the charge effect so separation proceeds on size alone. Agarose gel electrophoresis is routinely used in clinical laboratories to identify protein, including enzyme, ab-
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Figure 8. Variation of pore sire or with type of agarose 771
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Figure 6. S e w protein electrophoresis patterns showing variations of serum constituents indicative of disease states. Run in SeaKem ME agarose on GelBond film.
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Figure 7. DNA restriction digest electrophoresis in agarose of varying degrees of sieving. Samples: Hind I l l digest of A DNA and Hae 111 digest of QX174DNA. The three a g a r ~ ~ were e s run simultaneously within a framing gel in Tris/acetate/EDTA buffer. ph 7.8. for 18 h a t 30 V.
normalities in serum and plasma, as well as other biological fluids. A stained serum electrophoresis pattern is shown in Figure 6. Two critical procedures in recombinant DNA or genetic engineering techniques rely on agarwe gel electrophoresis: (1) separation and isolation of desired gene DNA fragments and (2) gene mapping (Figure 7). Because charge densities are essentially equal in DNA and restriction enzymecleaved fragments, all migrate according to size in electric fields stabilized by agarose. Using 0.1 to 2.5% agarose gels, resolution of about 0.1 to 900 kilobase pairs is possible. One can separate particles such as phages, viruses, and capsids, using even lower concentrations of agarose (0.035%) Serwer (1981). Frequently a scientist wishes to recover separated nucleic acids or fragments from elec-
trophoretic separations. The two low-gelling, low melting temperature hydroxyethyl agarose derivatives, SeaPlaque and SeaPrep agaroses, mentioned earlier (Figure 4) offer the flexibility of thermal disruption of the agarose matrix a t temveratures below the denaturation voint of the volynucleotides. Cross-linked polyacrylamide gel electrophoresis has freauentlv been used for molecular sieving of smaller moiecules. This material is produced by poiymerization in situ and can be difficult to form reproducibly. In contrast to agarose gels, it is also difficult to dry a run film one wishes to save, without cracking. Nochumson (1983) recently discovered that a nongelling olefinic agarose derivative, known as AcrylAide cross-linker, can be substituted for N,N'-methylenebisacrylamide to cross-link polyacrylamide gels, while maintaining resolving capacity. These gels can be readily oven dried to a coherent undistorted film even at 15% acrylamide-1% AcrylAide cross-linker concentrations, particularly on FMC's GelBond PAG fh. This simplifies both autoradiographic and fluorographic procedures for identifying gene products. A special form of electrophoresis, called isoelectric focusing or IEF, which takes advantage of the varying isoelectric points of amphoteric biopolymers and has been traditionally done on polyacrylamide gels, has now been adapted to specially-designed, negligibly charged agarose media. One such product, FMC's IsoGel agarose, is a mixture of a very low EEO agarose and a galactomannan which further supresses charge. Pharmacia designed a derivatized IEF agarose which has balanced anodal and cathodal EEO and therefore can be used for isoelectric focusing. In contrast to earlier beliefs, it has been shown by Nochumson et al. (1980) that the helical structure of agarose can be disrupted by hydroxyethylation resulting in increased sieving properties which are proportional to the degree of hydroxyethylation (Figure 8). A 4% SeaPrep agarose gel gives sieving properties about midway between 5% and 7.5% polyacrylamide, and B u z b and Chrambach (1982), have confirmed that gels with sieving properties equivalent to 10% polyacrylamide are possible. Recently, Coulson and Cook (1982), reported the use of agarose for two-dimensional (2D) electrophoresis, using IsoCel agarose for isoelectric focusing in the first dimension and a SeaPlaque, SeaPrep agarose gradient in the second. This development should allow examination of mixtures of large molecules which are totally excluded by polyacrylamide gels. Immunology. Applications of agarose in immunology for the detection of and study of various antigenic materials, particularly disease indicators, and their specific antibodies are so numerous that this presentation can barely scratch the surface. Antigens are defined as any substance not recognized as 'selF which will give rise to an immunological response. Antibodies (immunoglobulins) are defined as specific proteins formed by specialized animal cells. They are synthesized in response to an antigenic
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Ind. Eng. Chem. Rod. Res. Dew. Vd. 23. No. 1. 1984 Cell v i a b l i l y on SeaPllaque and A g a i
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Figure 9. Demmstmtion of antigen-antibcdy precipitin reactions in agarose gels.
stimulus and will combine specifically with that antigen to neutralize its threat to the animal. Many of these antigen-antibody complexes are insoluble, and if an antigen and antibody specific to that antigen diffuse separately through an agarose gel, the position where they come together will be marked by a cloudy or white so-called precipitin band marker (Figure 9). Because of the macroporosity of the agarose gel matrix (antibodies are large molecules). relative chemical neutrality, and high clarity, agarose is an ideal medium for immunological reactions. A number of immunological techniques, all employing agarose, have been developed which rely on visualized antigenllntibody interactions. These include gel diffusion radial immunodiffusion, immunoelectrophoresis, electroimmunodiffusion, counterelectrophoresis, etc. For further information, a basic immunology textbook and FMC's booklet, 'The Agarose Monograph" (Womer, 1983). can be consulted. Some of the immunobiological assays such as plaquing, to detect single cell antibody producers, hemolysis-in-gel, and migration inhibition factor (MIF) assays, have benefited from development of low-gelling, low melting temperature agarose products in which cells can be incorporated at temperatures so that they remain viable. Also, agarose particles to which antigens, antibodies, enzymes, or enzyme substrates are attached have enabled highly sensitive specific molecule or microorganism assays to be developed. Microorganism and Cell Culture. Agar has long been the standard medium for microorganism and cell culture. However, even the bacteriological-grade agar varies considerably from lot to lot and eo itains varying proportions of unknown entities, some of which are reported to be toxic to microorganisms and plant an;f animal cells. This leads to slower or no growth of sensitive cells and microorganisms. Agarose, because of its higher degree of purity and consistency, is finding increasing use by scientists for critical cultures. Antibody-producing hybridomas, after formation by fusion, have been found to reproduce more consistently on agarose Civin and Banquerigo (1983). One of the problems with agar, and this includes standard agarose preparations, is the relatively high gelling temperature. With the availability of lower gelling and melting temperature hydroxyethyl agarose derivatives, such as SeaF'laque and SeaF'rep agaroses, cells and other heat-labile substances can be incorporated into gelling media at considerably lower temperatures than before. In addition, these hydroxyethyl agarose derivatives seem to encourage cell growth, as demonstrated by Shillito (1982) (Figure 10). Chromatography. Columns of beaded agarose gel particles, sold under such tradenames as Sepharose (Pharmacia) and Bio-Gel A (Bio-Rad), serve as media for
Figure 10. Enhanced plant protoplast cloning efficiency in SeaPlaque agarose compared with agar media. (Courtesy of R. D.Shillib.)
molecular size separations because of the uniform effective pore size related to a particular concentration of agarose in the gel. Agarose is the preferred chromatographic medium for separations of molecules greater than 250000 daltons where minimal nonspecific binding to the medium and retention of biological activity of the molecules being separated are important. Affinity chromatography is a rapidly growing extension of agarose gel chromatography. In this technique, an antigen, antibody, enzyme, coenzyme, or substrate, etc. is bound physically or chemically to the agarose gel particle. These bound ligands interact specifically with molecules of particular physical and/or chemical conformation and thus remove them selectively from complex mixtures containing them. The specific molecules can subsequently be eluted by changing the composition, pH, and/or ionic strength of the eluant, thus effecting purification in a single step with high yields. Agarose may also be derivatized so that functional hydrophobic group ligands are available Hofstee (1973). Work by a number of investigators in the field of aga-based hydrophobic chromatography indicates promise for this versatile technique. More restricted pore media for chromatography have been developed (Uriel and Berges, 1966) by combining agarose with polyacrylamide. Such products are available commercially under the name Ultrogel (LKB). Finally, Schell and Ghetie (1972) developed a number of ionic agarose derivatives useful for chromatography. These and the aforementioned media have found considerable applications in the field of biotechnology, assuring a strong future for agarose beads and granules and their derivatives in separation and purification science. Immobilized Enzyme and Cells. Agarose immobilized cells and enzymes are important bioconverters. There have been many reports in the literature regarding the use of agarose gel films, particles, or beads to attach enzymes or encage cells and subsequently using them as bioconverters to transform one chemical to another. The interested investigator is urged to consult the literature for further specific information on this technique. One specific application recently described (Howell et al., 1982) which may have interesting consequences is the use of low gelling temperature SeaPrep agarose to encap sulate insulin-producingpancreatic Iselt of Langerhan cells
Ind. Eng. Chem. Prod, Res. Dev. 1984, 23, 21-27
for transplantation. Finally, the use of agarose to encapsulate activated charcoal and ion-exchange resins in agarose beads for hemowrfusion to detoxifv individuals in the case Of drug overdose looks promising (Holloway et 1979). ~. New biotechnology applications are continually being discovered for agarose and its derivatives. can be seen from the representative examples cited, agar and agarose, polysaccharides from seaweeds, or marine algae, are inOne Of partners in advancing the most exciting frontiers of science. Registry No. Agar, 9002-18-0; agarose, 9012-36-6.
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Coulson, S. E.; Cook, R. 8. “Electrophoresls ‘82”, Stathakos, D., Ed.; W. deGruyter and Co.: Berlln, 1982. D u c k w h , M.; Yaphe, w. cerbohyd. Res. 1971, 76, Guiselev, K. B. cerb.ohvd. R ~ S 1970. . 73, 247-256. Hotstw, B. H. J. Bloctiem. Res. co”.1973, 50, 751-757. Holloway, C. J.; Harstlck, K.; Brunner, 0. Int. J. A M . Org8ns 1979, 2 , n i-RR ””. Howell, S.L.; Ishaq,S.;Tyhwst, M. J . physw. 1982, 324, 20. Nochumson, S.; Cook, R. B.; Wllllams, K. W. Presentation at Ekctrophoresls Forum ‘80; Technlcal Unhrerslty, Munich, West Germany, 1980. Nochumson, s.; Qlbson, s. Bbrechniques 1983, 7 , 18-20. Schell. H. D.; ohetie, V. Rev. Roum. Blochim. 1972, 9 , 165-177. Serwer, P. Anal. Blochem. 1981. 172, 351-356. Shllllto, R. D., Frledrlch Mlescher-Institut, Basel, Switzerland, personal c0t-t-tmunlcatlon, 1982. Urlel, J.; Berges. J. C. R. Aced. Scl. farls 1986, 262, 164-167. Womer, M. C., Ed., ”The Agarose Monograph”, Marlne Collolds Dlvlsion, FMC Corporation, Rockland. ME, 1982. “ I
Literature Cited
Received for review April 20, 1983 Revised manuscript received August 30, 1983 Accepted October 3, 1983
Buds, 2.; Chrambach, A. Electrophoresis 1982, 3, 130-134. CMn. C. I.; Banquerlgo, M. L., Johns Hopklns Unhrerslty. Baltimore, MD, personal communication, 1983.
I I I. Symposium on Synthetic and Petroleum Based Lubricants B. L. Cupples, Chairman 183rd National Meeting of the American Chemical Society Las Vegas, Nevada, March 1982 (Continued from June 1983 Issue)
Some Synergistic Antioxidants for Synthetic Lubricants Tal S. Chao,’ David A. Hutchison, and Manley Kjonaas ARC0 Petroleum Products Company, Dlviskm of Atlantlc Richflekl Company, Harvey, Illlnols 60426
Oxidation stability is a major requirement for synthetic lubricants, especially for those used in aircraft gas turbine engines, owing to the high-temperature oxidative environment. This paper describes some antioxidant systems which we found to be highly effective in improving the oxidation stability of synthetic lubricants. These systems contain a primary antioxidant, usually an aromatic monoamine, and a synergistic antioxidant. The latter is a compound which, when added in small concentrationsto the primary antioxidant, greatly improves its effectiveness. Synergistic antioxidants discussed will include alkali metal compounds, heterocyclic amines, aromatic diamines, hydroxybenzophenones, and certain sulfur and phosphorus compounds. Discussions will also be made of a bench test used for screening antioxidants and possible mechanisms of action of these antioxidants.
Introduction Oxidation stability is a major requirement for all lubricants. It is even more important for synthetic lubricants used in aircraft gas turbine engines, owing to the high oil temperature and air exposure. Two basic means are available to improve oxidation stability of synthetic lubricants: choice of base fluid and use of antioxidants. It is well-known that fluids such as polyphenyl ethers are much more oxidatively stable than fluids such as dibasic acid esters. Discussions of the effect of the nature of the C-H bond on oxidation stability have been made in our previous papers (Chao et al., 1979, 1983) and by other authors such as Ingold (1961a,b) and Sniegoski (1963). However, the choice of base stock is severely limited by cost and other considerations such as volatility, flow characteristics, etc. When the choice of base fluid has been made, use of a better antioxidant system is the only avenue available to improve the oxidation stability of the lubricant. This paper describes some of the antioxidant systems which were found to be especially effective for ester type 0196-432118411223-0021$01.50/0
synthetic lubricants. The esters used included those based on dibasic acids, neopentylpolyols, and complex esters. The antioxidant system discussed includes one or two primary antioxidants and a synergistic antioxidant. The primary antioxidants used included N-phenyl-anaphthylamine (PANA), p,p’-dioctyldiphenylamine (DODPA), and other aromatic amines. Synergistic antioxidants which were found to be very proficient in improving the effectiveness of the primary antioxidants include alkali metal compounds, aromatic diamines, heterocyclic amines,hydroxybenzophenones, and certain sulfurand phosphorus-containing compounds. Experimental Section Preparation of Alkali Metal Compounds. Alkali metal salts of partial esters of ethylenediaminetetracetic acid (EDTA) were prepared either by partial esterification of EDTA with alcohol, followed by neutralization with Na2C03or K2C03,or by the partial saponification of the tetraester, with NaOH or KOH. The latter method gave better control and provided relatively pure products. In 0 1984 American Chemical Society
