Antigens and Enzymes Made Insoluble by Entrapping
`Them into Lattices of Synthetic Polymers
`Biologically active, insoluble products have been prepared from
`Abstract.
`soluble antigens or enzymes by entrapping them into the lattice of an insoluble,
`highly cross-linked synthetic polymer as it forms. Antigens, made insoluble,
`removed quantitatively certain or all antibiotics from complex mixtures. Seven
`enzymes so treated are described.
`Soluble
`previously
`antigens
`have
`been rendered insoluble either by cou-
`pling the proteins with diazotized cel-
`(1 )
`lulose derivatives
`or by linking
`them to the acid chloride of a carboxyl-
`ated
`(2).
`Also,
`resin
`ion-exchange
`insoluble forms of amylase, pepsin, car-
`boxypeptidase, and ribonuclease have
`been obtained by coupling with diazo-
`tized polyaminostyrene (3), and insol-
`uble
`was obtained
`by
`ribonuclease
`absorption on a Dowex-50 cation-
`exchange resin
`(4). Tryspin (5) and
`papain (6) were transformed into in-
`soluble derivatives by the introduction
`of polytyrosyl peptide side chains. This
`reaction is not applicable without con-
`siderable modifications to other proteins,
`or protein mixtures. Insoluble proteins
`obtained by coupling with diazotized
`polymers or by attachment to ion-ex-
`partial
`change resins
`suffered
`either
`denaturation during these reactions or
`exhibited partial reversibility of the ab-
`sorption, under certain conditions (5).
`We have obtained biologically active,
`insoluble forms of antigens, enzymes,
`material
`macromolecular
`and
`other
`(for example, amylopectin), by me-
`chanically entrapping the soluble ma-
`cromolecular product into the lattice
`of a highly cross-linked synthetic poly-
`mer by polymerizing certain synthetic
`monomers in aqueous solution in the
`presence of the biologically active ma-
`cromolecular substance to be embedded.
`which
`polyacrylamide,
`Cross-linked
`in an aqueous medium
`polymerizes
`and which has been used as a support-
`ing medium for zone electrophoresis
`(7), is eminently suited for this pur-
`pose. We have used two methods for
`rendering proteins or other macromo-
`lecular biologically active material in-
`soluble, depending on the purpose for
`which the resulting products are to be
`used. For rendering proteins or other
`antigens insoluble (procedure I) 50 mg
`of antigen are added to 200 mg of
`acrylamide, 120 mg of N,N' methylene-
`bisacrylamide, 5.6 mg of tetramethyl-
`ethylenediamine, 0.5 mg of Al NH4-
`(SO4)2' 12 H20, and 1 ml of (1M) tris
`1 ml
`buffer, pH 8.6, all diluted to
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`with water. At the last, 2.5 mg of
`potassium persulfate
`are added. For
`rendering enzymes insoluble (procedure
`II), 2 mg of the enzyme are added to
`a mixture similar to the one described
`for procedure I except that the acryl-
`amide is omitted and the amount of
`the potassium persulfate added is 5 mg.
`In both procedures, the mixture is
`kept for 1 hour at 35°C without agita-
`tion after the polymerization catalyst
`(potassium persulfate) has been added.
`The insoluble synthetic polymer thus
`formed is dispersed mechanically, cen-
`trifuged for 30 minutes in a Sorvall
`centrifuge, model RC-2, rotor SS-34,
`at 17,000 rev/min (34,000g), and is
`washed by mixing it with 20 ml water
`and subsequent centrifugation. A total
`of three washings for procedure I and
`16 washings for procedure II are neces-
`sary.
`
`Table 1. Activity of crystalline enzyme (9)
`entrapped in the synthetic polymer prepara-
`tion and the residual activity remaining in
`solution after the removal of the synthetic
`is
`the
`polymer.
`The "entrapped"
`activity
`percentage of total amount of enzyme pres-
`ent during the polymerization reaction. The
`is measured by first in-
`"residual"
`activity
`cubating the substrate at 250 to 350 C with
`insoluble enzyme for periods ranging from 25
`to 180 minutes, cooling the mixture to 0°C,
`and removing the insoluble enzyme by cen-
`trifugation and subsequent filtration, and in-
`cubating the remaining solution at 250
`to
`350C for 18 to 22 hours. Residual enzyme
`activity
`activity
`expressed
`is
`in
`percent
`per unit of time before centrifugation.
`Activity
`Residual in
`supernatant
`solution
`(%)
`4-5.5 t
`Trypsin *
`0.65
`0.77
`a-Chymotrypsin *
`4.5
`Papain *
`0.57
`3.4-6 t
`ca-Amylase t §
`0.7
`1.9
`3-Amylase
`0.3
`6.55
`§
`0.89
`4.6
`Ribonuclease 1
`0.52
`4.2
`Aldolase * *
`digestion method
`casein
`* Determined by the
`t Two separate experiments.
`t From
`(10).
`§ Determined by the reducto-
`hog pancreas.
`metric method with dinitrosalicylic acid reagent
`II From sweet potatoes.
`I From beef
`(11).
`pancreas; activity determined by the spectrophoto-
`** From rabbit mus-
`metric method (12, 13).
`cle; activity determined by the method of Taylor
`et al. (14).
`
`Entrapped
`(%)
`
`Enzyme
`
`SCIENCE, VOL. 142
`
`awaits definitive biochemical and bio-
`logical studies.
`The control diet at Darrah Springs,
`diet
`8, contained 19 percent cotton-
`In contrast, diet 9 (liver
`seed meal.
`and cottonseed meal)
`contained 37
`percent cottonseed meal when calcu-
`lated on a dry-weight basis. Yet at the
`end of 10 months, 40 out of 50 trout
`in the group fed on diet 8 had gross
`hepatomas, but neither gross nor mi-
`croscopic tumors were found in the 50
`trout sampled from the group fed on
`At the end of 23 months,
`diet
`9.
`among 65 fish fed on diet 9 only 3
`These ob-
`gross tumors were found.
`servations suggest that liver markedly
`the hepatoma-inducing effect
`inhibits
`of the cottonseed meal, but does not
`completely suppress it. Nakahara et al.
`(10) have demonstrated the inhibitive
`effect of a diet containing liver on
`hepatoma induction by butter yellow
`(dimethyl-amino-azobenzene)
`rats,
`in
`and a similar effect may be operating
`in this instance.
`The results obtained with diets 2 and
`4 obscure the significance of the four
`microscopic tumors found in diet
`6.
`With the omission of wheat middlings
`in diet 4, the incidence of hepatomas
`was no lower than in the control group;
`with diet 2, no hepatomas were found,
`although the diet included 25 percent
`wheat middlings.
`For these reasons,
`we believe that this component played
`no significant role in the outbreak of
`hepatomas in California during 1960,
`when the incidence of tumors in very
`young fish was extremely high (11).
`HAROLD WOLF
`California Department of Fish
`and Game, Sacramento
`
`E. W. JACKSON
`California Department of Public
`Health, Berkeley
`
`References and Notes
`1. A. Haddow and J. Blake, J. Pathol. Bacteriol.
`36, 41 (1933).
`2. G. Cudkowicz and C. Scolari, Tutmori 41, 524
`(1935).
`3. R. F. Nigrelli, Trans. Amer. Fisheries Soc.
`83, 262 (1954).
`4. J. H. Wales, personal communication (1960).
`5. J. Ellis, personal communication (1960).
`6. R. R. Rucker, W. T. Yasutake, H. Wolf,
`Progressiv,e Fish Culturist 23 (1961).
`7. E. M. Wood and C. P. Larson, Arch. Pathol.
`71, 471 (1961).
`8. W. C. Hueper and W. W. Payne, J. Natl.
`Cancer Inst. 27, 1123 (1961).
`9. "Summary of Trout Hepatoma Survey," U.S.
`Fish and Wildlife Service, Bureau of Sport
`Fish and Wildlife, Sept. (1960).
`10. W. Nakahara, K. Mon, T. Fujiwara, Gann
`33, 406 (1939).
`11. This investigation was supported in part by
`Public Health Service grant No. C-5924 from
`the National Cancer Institute.
`14 August 1963
`
`678
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`I
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`A
`B
`C
`Fig. 1. Antigen-antibody interactions by the agar plate diffusion technique of Ouchter-
`lony. All center wells (in A, B, and C) contain rabbit antiserum specific for whole
`human serum and for crystalline bovine serum albumin. Center well in A, antiserum
`treated with insoluble human serum protein (prepared from pooled whole human
`serum). Center well in B, antiserum untreated (control). Center well in C, antiserum
`treated with insoluble bovine albumin (prepared from crystallized bovine serum albu-
`min). The outside wells have the same arrangement in A, B, and C, that is, upper and
`lower left, bovine serum albumin (157 ug per ml); upper right: whole human serum
`(1:128 dilution); lower right, mixture of whole human serum (1:128) with bovine
`albumin (157 pg per ml).
`
`(see Table 1). After centrifugation and
`filtration, more than 99 percent of the
`enzyme activity had been removed from
`the mixture in all cases (residual ac-
`tivity was less than one percent of the
`activity before centrifugation).
`The
`rendered
`enzymes
`insoluble
`maintained their activity when stored
`to 4°C. A ribonuclease prepara-
`at O0
`tion, for instance, exhibited 90 percent
`of the original activity after one month.
`There was no indication that the en-
`properties
`zymatic
`of
`the
`insoluble
`products were in any way different from
`those of the corresponding soluble ma-
`terials and, in particular, the pH opti-
`ma of activity were unchanged. In the
`tests made, contact with the substrate
`did not release even traces of any of
`the entrapped enzymes.
`Freshly prepared
`insoluble papain
`was active in the absence of added
`sulfhydryl agents or of cyanide, and
`was not activated by any of these mate-
`rials;
`this
`is comparable to the pa-
`pain derivative described by Cebra et
`al. (6). Our insoluble papain prepara-
`tion completely lost its enzyme activity
`when stored for 3 days at 00 to 4°C
`(air was not excluded); however, it
`could be reactivated by cysteine.
`Biological activity of antigens or en-
`zymes requires contact with antibodies
`or substrates,
`respectively, but most
`antibodies,
`and many enzyme sub-
`strates, are macromolecular substances
`and hence are unable to penetrate into
`the particles of the cross-linked poly-
`the same reason that
`mer for
`the
`trapped antigens or enzymes cannot
`get out. It appears likely therefore that,
`
`while the entrapped antigen or enzyme
`may be located both inside and near
`the surface of the insoluble particles,
`only the latter portion exhibits biologi-
`cal activity. Actually, considerably more
`than 2 to 6 percent of the original en-
`zyme (or antigen) may be entrapped
`(Table 1) but may not exhibit biologi-
`activity
`cal
`for
`of
`steric
`reasons
`hindrance (8).
`
`PETER BERNFELD
`J. WAN
`
`Bio-Research Institute,
`Cambridge, Massachusetts
`
`References and Notes
`1. D. H. Campbell, E. Luescher, L. S. Lerman,
`Proc. Natl. Acad. Sci. U.S. 37, 575 (1951).
`2. H. C. Isliker, Ann. N.Y. Acad. Sci. 57, 225
`(1953).
`3. N. Grubhofer and L. Schleith, Naturwissen-
`schaften 40, 508 (1953).
`4. L. B. Barnett and H. B. Bull, Blochim. Bio-
`phys. Acta 36, 244 (1959).
`5. A. Bar-Eli and E. Katchalski, Nature 188,
`856 (1960).
`6. J.
`Cebra, D. Givol, H. I.
`J.
`Silman,
`E.
`Katchalski, J. Biol. Chem. 236, 1720 (1961).
`7. S. Raymond and L. Weintraub, Science 130,
`711 (1959).
`8. Supported in part by grant CA-03852 from the
`National Institutes of Health, by grant P-249
`from the American Cancer Society, and by
`a grant from the National Science Founda-
`tion.
`9. Aldolase was purchased from Mann Research
`Laboratories;
`all
`other enzymes were pur-
`chased from Worthington Biochemical Corp.
`Insoluble hyaluronidase from a bovine testes
`preparation, with 300 USP units per milli-
`gram, has also been obtained.
`10. M. Kunitz, J. Gen. Physiol. 30, 291 (1947).
`11. G. Noelting and P.
`Bernfeld, Helv. Chim.
`Acta 31, 286 (1948); P. Bernfeld, in Methods
`in Enzymology, S. P. Colowick and N. 0.
`Kaplan, Eds. (Academic Press, New York,
`1955), vol. 1, p. 149.
`12. M. Kunitz, J. Biol. Chem. 164, 563 (1946).
`13. C. B. Anfinsen, R. R. Redfield. W. L. Choate,
`J. Page, W. R. Carroll, ibid. 207, 201 (1954).
`14. J. F. Taylor, A. A. Green, G. T. Cori, ibid.
`173, 591 (1948).
`9 September 1963
`
`679
`
`The first procedure (I) yields a gel-
`like, insoluble synthetic polymer which
`can be easily and completely removed
`from the suspension by centrifugation;
`particular mechanical
`because of its
`properties, this material is not suitable,
`however, for accurately measuring out
`or pipetting samples of its suspension.
`The second procedure (II) results in a
`flocculent product which lends itself
`well to quantitative measurement, but
`separation of the solid from the liquid
`phase is more difficult.
`To use insoluble antigens for the
`absorption of antibodies, the solid ma-
`terial obtained after the last washing
`(procedure I) is mixed with 1 ml of
`an antiserum (for example, rabbit anti-
`serum against whole human serum),
`and the suspension is kept under con-
`stant agitation (by means of a Boerner
`for 2 hours at
`oscillating platform)
`room temperature. The insoluble anti-
`gen is then removed by centrifugation
`at 34,000g, and the protein concentra-
`tion of the remaining solution is ad-
`justed to that of the antiserum before
`treatment by lyophilizing and dissolving
`the residue in water.
`The photographs of the antibody-
`antigen reactions in Fig. I show that
`antibodies against a mixture of a large
`number of antigens (whole human se-
`rum) can be quantitatively removed by
`this method (Fig 1A, upper and lower
`right), while the antibody against an
`additional antigen, which was absent
`from the mixture of insoluble antigens
`used (bovine serum albumin), is totally
`unaffected (Fig. 1A, upper and lower
`Inversely, a single
`left, lower right).
`antibody can be selectively removed
`from a complex mixture of antibodies
`(against whole human serum and bo-
`vine serum albumin) when its pure
`antigen (crystalline bovine serum al-
`bumin) is available (Fig. 1C).
`insoluble
`with
`For reactions
`en-
`zymes, a portion of the suspension of
`insoluble enzyme (procedure
`is
`II)
`mixed with the appropriate substrate
`and buffer solutions, and the mixture
`is incubated under continuous agitation
`by means of a Boerner oscillating plat-
`form. The enzyme reaction is arrested
`by centrifugation of the mixture at
`34,000g and subsequent filtration of the
`supernatant solution through a What-
`man No. 1 filter.
`Between 2 and 6 percent of the en-
`zyme activity, present during the poly-
`merization reaction, appeared in the
`exhaustively washed synthetic polymer
`8 NOVEMBER 1963
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`Antigens and Enzymes Made Insoluble by Entrapping Them into Lattices of Synthetic
`Polymers
`Peter Bernfeld and J. Wan
`
`142
`Science 
` (3593), 678-679.
`DOI: 10.1126/science.142.3593.678
`
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