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Preparation of electroendosmosis-free agarose gel and exemplification of its use in crossed immunoelectrophoresis
Preparation and
of Electroendosmosis-Free Agarose Exemplification of Its Use in Crossed
Gel
lmmunoelectrophoresis ANDERS
GRUBB
The method Preparation of electroendosmosis-free agarose is dcscribcd. is based upon the vovalcnt honding to agnrow of a small numhrr of poaiI irely charged groups which countcrhalance thp elec:trocnclosnlosis-llro(~~lcirl~ effect of the negatively charged groups present in commercial agarosr. It is shown how such clectroendosmosis-free agarow gel can he ~ombinrd will) advantage with polyacrylamidr pls in vrosscd ilnm~lnorlrc~tro~,hor~~i~.
Crossed immunoclectrophor~~sis can often be used with advantage instead of the classical immunoelectrophorcsis of Williams and Grabar (l), since it has a higher resolving power, it is sellliquantitativc, and it requires only a few hours (2,3). In crossed imrnunoclcctroI,horesis a first zone electrophorctic separation in an agarose gel is followed by a second electrophoresis of the separated antigens into agarose gel containing antibodies, with the electrical field perpendicular to the one used in the first zone electrophoretic separation. The resolving power of the technique is, of course, dependent on, above all, the resolving power of the first step. If the zone electrophoretic separation in agarose gel could be replaced by other common procedures for separation of macromolecules, such as polyacrylamide gel electrophoresis or isoelectric focusing in polyacrylamide gel, it would notably imrease the versatility of crossed immunoelectrophoresis. The main obstacle to replacement of the agarose gel separation by separation in polyacrylamide gel is the difference in electroendosmosis bctwecn the agarosc gel containing the antibodies and the polyacrylamide gel containing the separated ant,igens. The obstacle would be overcome if an agarose gel could be produced which, like polyacrylamidc gel, had very little or no electroendosmosis. Agar, from which agarose is refined, has long been regarded as a mixture of the two polysaccharides agarosc and agaropectin (4). The agarose has been considered a charge-free polymer and hence considered Copyright .U rights
582 @ 1973 by Academic Prrss. Inc. of reproduction in any form rwzrwd.
to form electroendosntosis-free gels when dissolved in buffers, Fvhilc agaropectin has hecn regarded a.q a negatively charged polysaccharidc and hence considered to cause the elertroendosmotic flow against the cathode in agur gels. This concept of the structurc~ of agar led to several attempts to purify the completely uncttargcd agarosc from agar (5-8~‘~ hut, although low-charged polysacchnridc~s ww ~~rodurccl, no completcl> uncharged agarosc was ohtaincd. The reason why thcsc attempts at purifying agarose wrc unsuccessful has Iwtln csplaincvl hy wwnt invcstigwtions of thv structure of agar according to nltich agar consists of a hut family of ~~olys:tccltarides with similar ni:~~rottiolcc~ulnr structures with continuously varying degrees of aciclic suhstitrtcnts and with virtually no completely unchargt~tl mol~~~tlcs (8,9 1 Thcrc thus swms to 1)~ only two ways in whic.11 clectroeticlostiiosis-fr~~~~ ngarow (‘ati tw prorluwrl. One of them is by c~lic~rrii~al rctiioval of all acitlic sut~stitucnts of thcb ha\-(5 Iwcti taken 1IO,1 1 ), agarose. Important steps in this dirwtion agarosc ,swttis to have t~ecn hut no completely elcctrocti~lost~iosis-frrc obtained. The otltcr l)ossil)lr way to l)roduw (:lcrtrocntlosmosis-free agarose is to introrlucc into tlw polysacrtiarid~~ :I .stn:tll amount of lwsit,ively charged suhstituetits which couiitcrbalan~c~ the olt~ctrortitlostiiosisI)roducing effect of tlw ncgativcly cltargcd groups I)rcacnt. in commercial agarow. This paler clcwrihw how such basic substiturttts can tw itttrodrtced into agarosc without destroying its gelling ability. Tt also demonstrates how tltc corresponding ctcrtroc~ndosmosis-fr(~(l gel can tw utilized in crossed itiiuiuttorlcrtrol~horcsis with :I iwl\-ncrylamidc gel separation in the first dimension. MATERIALS
hgarow (A-45, Ittclut~iosc, hatch 3033) was lntrchasrd from I,‘Indus-tric~ Biologiqtw Fran~aiw S.A., (knnevillicrs, Franw, Ikstrnn 40 iMT1’ 40,000, unrhargrd I from Pharmacin Fittp C’hcmirals AR, a highI> purified quality :wrylnntidr, No. 5.521S, from Hasttnan Kodak C’o.. ~V,N,~\;‘.~\~‘,-tctr:ttitcth~lct~iyteii~~~li:ttiiiti~~ :Illd .~,.\-‘-mc~tltplencl~isa~rytamide from BL)H C’ltcwicals I,tcl. ~\~..~\r’-t~~cthylcIlchis:~~ryla~~~i(le was wcrystallized once from chloroform. Atnpltolytc~ solutions wew bought from I,KB Products and cyanogen hromitle from Fluka 4.G. All other chemicals except the 1primary qua&nary diamine described hctow, ww of analytical grad(J obtained from commercial sourws. Preparation. oj i.%aminoeth~yl~t~iNlethylanlvnonilrnl bromide h!ydrobromide. 1 equivalent of anhydrous trimethylamine was conducted into nitrobenzenc containing 1 equivalent 1,2-dihromoethane during continuous stirring at room temperature. The resulting (2-hromorthyl) trimethylammonium bromide was then extracted into distilled water, which was
584
ANDERS
GRUBB
subsequently evaporated. The salt was t,hcn recrystallized from ethanol and dissolved in 10 equivalents of concentrated aqueous ammonia. After 6 days at room temperature the solution was taken to dryness by evaporation. Small amounts of yellow-colored byproducts were dissolved in a small volume of hot ethanol. The product obtained was a white cryst,alline powder. The purity of the product was tested by thin-layer chromatography on aluminium oxide coated and on silica gel coated plates (developed with n-butanol/glacial acetic acid/water, 34/34/132, v/v) as well as by high-voltage leaper-clcctrophorcsis in a phosphate buffer of pH 12.5. Staining with either ninhydrin or dipicrylamin (12) revealed only one spot in the chromatograms and in the electropherogram. During the latt’er part of this work, the intermediary product (2-bromoethyl) trimethylammonium bromide became available from Schuchardt A.G. METHODS
Introduction of positizlely charged gwups into agarose molecules. Agarose was dissolvtd to a concentration of 0.337% in boiling distilled water, and the solution was allowed to cool to room temperature. A viscous solution was formed. The pH of the solution was adjusted to 11.0 with 0.2 M sodium hydroxide. Immediately thereafter, a freshly prepared water solution of cyanogen bromide wa#s added to the agarose solution during vigorous stirring. The stirring was continued for the next 10 min, and the pH was kept, at 11.0 by small additions of the sodium hydroxide solution and the temperature at about 20°C by the addition of crushed ice. Immediately after this period, a 2.0 M solution of (2aminoethyl) trimcthylammonium bromide in water adjusted to pH 10.0 was added. The mixture was then left, at, 4°C overnight. It was then centrifuged for 15 min at lO,OOOg, which resulted in the formation of a gel at the bottom of the centrifuge tube. The supernatant was discarded, the gel shaken with dist.illed water and then centrifuged down again. The washing procedure was performed three times. The washed gel was then frozen and thercaftcr allowed t’o t’haw in a nylon net, whereby the gel lost at least 90% of its water. Finally, t’he gel was freeze-dried. Determination of electroendosmosis. The freeze-dried agarose derivative was dissolved to a concentration of 17% in a buffer solution’ on a boiling water bath, and then a 1-mm-t’hick gel was cast on a microscope ‘To avoid alkaline hydrolysis of the agarose derivative when a gel was to be prepared in a glycine buffer of pH 10.3, the derivative was dissolved in distilled water. the solution cooled to about 50°C. and then a concentrated buffer WBS added immedistelqbefore the gel was cast.
slide. This was then J~lacctl in a water-cooled elcctrophoresis apparatus with a tightly fitting lid (13) and connected with the electrode vessels by means of gel bridges cast, with the same gel as the one on the slide. A slit was filled with a solution of d&ran 40 itt the buffer used for electrophoresis, and a pot&in1 graclic~nt of I5 V;/cm was applied for 3 hr. Thereafter the slide was immersed in a (Ir,r;trntt-1)rr~~i])itatittg solution of acet.ic acid, cthsttol. and water (,5/70/‘25, Y, Y I (14). The vlect,roendosmosis was then cnlculatetl from tlicl clktattce tr:tvcIet] by the uncharged dextran, the electro])ltorcGs t.imc, :LINI the J)0t8ential gradient. Isoelectric Joc~~Gq in pol~yncryltrvride qel. The ])roccdurc described by .Jeppsson and Rergluntl (1.5,) K:~S uac~l. Polyacrylnvlitle slab elect?,ophclresis. This was performed ]y preparing a l.O-mm-thick 5:/r J’ol~--:trrylnltticl(~ gel on Ixtrt of a glass-plate and the suhsquettt casting of an cl(,~trocllc]o~;moai8-free agnroac gel on the free part of the glass-plate. ~~l)l)lic~:ttiott slits were ]~o(lu~d in the ngarose gel by inserting a mold nith 15 rc~ctnngular teeth in the agarose solution and removing it. nftt>r the, Iattcr hart sctt. The two types of gels were prepared using thv same barbital ])uffcr 1’0.07531, pH 8.6 1, and the electro])ltorcsis was perfortticd horizotttally at 20 FTi’ct~t in a simple water-cooled elcct,ropltorcsis ap]):tratus (13 i The gchl5were connected with the rlcctrotle vessels containing the lrur],ital brtfi’cr by tneans of thick electroendosmosis-frc~c~:tg:tro~c~go] IJritlgW;. This simple technique for polyacrylamitlc slab electrophorc~ris iLq (lcsc~rihcclin clotails elsewhere (16).
When increasing nmount~ of cyattogcln bronti~l~~wcrc used in the introtluction of positively chargccl grottl~ ittto :q:trose. thcx c,lcc,trocndosmclsi::
of the resulting gels was influenced in the way depicted in Fig. 1. The final concentration of (2-aminoethyl) trimethylammonium bromide was kept at 0.06 M in these experiments. The electroendosmosis was tested in 1% gels in either barbital buffer (pH 8.6; 0.075 MI or glycine buffer (pH 10.3; 0.06 M I, il’o significant difference in electroentlosmosis was found between t,he gels in the two different buffers. It can be seen that the cathodal elcctroenclosmosis of unaltered agarose diminished and was converted to an anodal one as the amount of cyanogcn bromide increased. The anodal elcctrocndosmosis increased with the amount of cyanogen bromide added until an agarosc product insoluble in boiling dist,illed water was obtained by an addit’ion of about 1.5 g cyanogen bromide/g agarose. When a fixed amount of cyanogen bromide (0.3 g/g agarosel was used to introduce positively charged groups into agarose but the final concentration of (2-aminoethyl) trirliethylarllrnonium bromide was varied, the electroendosmosis of the derived gels varied as shown in Fig. 2. The electroendosmosis was tested in l$Z gels in the two buffers mentioned above. Gels prepared in the two buffers had the same clectroendosmosis. An increase in the concentration of (2-aminoethyl) trimethylammoniumbromide caused t,he cathodal electroendosmosis of unaltered agarosc to diminish and to convert to an anodal one. To prepare an electrocndosmosis-free agarosc gel, an agarosc clcrivative was produced that, had so many positively charged groups t,hat the corresponding gel posacssed nnodal clcctroendosmosis. This was accomplished as described in Methods with the addition of 0.3 g cyanogcn bromide/g agarose and with a final concentration of 0.15 M (2-aminoELECTROEN)OSMOSlS
(lb5cn+&‘)
+0.4 1
CNBr-ADDITION
-O.& 0
0.1
1. The electroendosmosis increasing amounts of cyanogen aminoethyl) trimethylammonium c~lectroendosmosis and negative FIG.
0.2
a3
0.4
(g/g 0.5
at pH 8.6 of gels prepared by bromide and constant amounts bromide. Positive numbers numbers cathodal ektroendosmosis.
agarose) 0.6
a reaction of agarose designate
between and (2anodal
ELECTROENDOSMOSIS
(165cm%‘)
-0.8
CONCENTRATION 0
0.1
a2
al
61)
a4
2. Tlrc clcctroendoamosis at pH 5.6 of gels prepnred by 3 wattion hrtween increasing amounts of (2-;mGnwthyl) trin~~th-lxmmoniunl~~l~~n~noni~~~~ bromide and constnni amounts of agnrose and cycmogcn hromitlc~. Positirr numhrrs dcaignnte nnodal elwt,rorndosmosis nnd nrg:ltivc nrlmhrw c3ihod:rl c~lr~troenclorn~o~i~. FIG.
ethyl 1trimcth\;l:ttntnotlittttl t)rotttide. E;l(~ctrocntlosmosiu-free agarose was obtaiwd by tnising an approl~riste volume of a solution of the agarose clerivative with nnodnl clectroetttlostllosis with a solution of commercial agnrosc (,witli cathodal electrocndostnosis) . -4nalyt,ic gel clcctrophotwia of scvcrnl human plaamns with the use of a 1% elcctrocttclostnosis-free agarosc gel in barbital buffer (0.075 M ; pH 8.6) produced the protein ltattcrn s in Fig. 3A. No protein adsorption was observed dcspitc t’he prcscncc of a small number of positively charged groups in the gel. If ltuman plasma containing haptoglobin of type 2-l or 2-2 is subjccted to Itolyacrylatnide slat) elcctrophoresis, tltc polymers of the haptoglobin types will separate mainly according to their molecular size. If the lwlpmers arc allowed to tnigrate ink0 elcctroetidost~~osis-free agarose containing anti-ltal~toglot~iti serum in crossed irnmunoelectro1)horesis. tltc individual polymers will be viaualizcd as alto~~n in Figs. 4 and 5. It Call h sect1 that the series of haptoglobin 2-l ltolymers contain at least 9 diffrrertt tttoleculr~ and t,hat at least I.5 different, haptoglobin 2-2 polymers exist. If ironsaturated human plasma is subjected to isoelectric focusing between pH 4 and 6 in I~olyncrylamide gel, numerous protein bantls will appear. At least 5 of thcw rcprcsettt diffcrcnt molecular forms of transfm’rill its ShOWJl by the rroswd irnnitttioclectrol,hor~~sis of Fig. 6.
FIG;. 3. Analytic gei elcctrophoresis of sis different human plasmas (or sera) with the use of (A) a 1% electrocndosmosis-free agarose gel or (B) a 1% commercial unaltered ngarosr gel in barbital buffer (0.075 M; JIH S.6). The voltage 20 V/cm was applied for 50 min, and the proteins stained with Amide Black B.
“^
590
ANDERS
GRUBB
human plasma. 7’1~ E’rc:. 6. Crossed inlmunoelectroplloresis of ken-snturatc,d tirst, elrctrophorctic step was performed by isoelectric focusing between pH 4 and 6 of plasma in polyacqlamide gr,l and the second by rlrctrophorrais in 1% cltctrorndoamosis-frer, agarosc> .~rl in barbital buffer (0.075 M: pH 8.6) containing antitixnsfrrrin senlm.
DISCUSSION
The incorporation of positively charged groups into agarose related here is based upon the reaction, previously described, between agarose and cyanogen bromide which results in an “activated” agarose that readily reacts with primary aliphatic amines with the production of water-irisolu~)lt~ agarosc products (17-20) In this study, it was found possible to obtain :I posit,ively c~hargccl agarosc clerivatiw that was soluble in water solutions and that retainrd itu gelling ability by the
592
ANDERS
GRI’BH
very acidic proteins to the gel and, second, the ionizable groups of the gel render it unsuitable as a stabilizing medium in isoelectric focusing. ACIiSOWLEDGMENT This University
investigation of Lund.
was
supported
by
n
granf-
from
Ihe
Medical
Faculty.
REFER,ENCES 1. GRABAR, P.. AND WILLIAMS, C. A. (1953) Biochim. Biophys. Acta 10, 193. 2. LAURELL, C.-B. (1965) Anal. Biochern. 10, 358. 3. GANROT. P. 0. (1972) Scctnd. J. c’lin. Lnb. Z?luest. 29 (Suppl. 124), 39. 4. ARAKI, C. (1965) in Proc. Fifth Int. Seaweed Symp. (Young, E. G.. and McLachlan, J. L., eds.), p. 3, Pergnmon Press, Oxford. 5. HJERTBN, S. (1962) B&him. Biophys. Acta 62, 445. 6. JOHANSSON. B. G., AND STEKFLO. J. (1971) Anal. Biochem. 40, 232. 7. HJERTBN, S. (1971) J. Chromutogr.61, 73. 8. DUCKWORTII, M., AXD YAPHE, ‘11:. (1971) Cnrbohyd. Res. 16, 189. 9. IZUMI, Ii. (1971) Carbohyd. Res. 17, 227. 10. PORATH, J., JANSON, J.-C., AK~ I,.%, T. (1971) J. C’h~n~tog~. 60, 167. 11. LKs. T. (1972) J. Chyomntogr. 66, 347. 12. STAHL, E.. AND SCHORN, P. J. (1967) irl Dii~~nac~hirht-C1~o~~~~togr;~l~hic~ (StallI, ‘E.. ed.), 1,. 474, Springer-Verl:lg. Berlin. 13. JOHANSSON. I:. G. (1972) fkc~u/. J. ('/in. Lab. znzwt. 29 (SUI,II~. 124), 7. 14. WIEME, R. J. (1965) in Agnr C;cll Elcctrophoresis (Wk~mc. R. J.. ed.), 1,. 99. Elsevirr. Amsterdam. 15. JWPSYOS, J.-O., AKD HERGLUKIL S. (1972) Cliu. C’hirn. dctcc 40, 153. 16. GWBB, A. (1973) Pmt. Biol. Fl~Lls 21, in prws. 17. Ax$N, R., PORATH, J.. ASI) F:RNBAC-K. S. (1967) .Vntvre (Lor~ion) 214, 1302. 18. PORATH, J., Ask. R., AND ERSBACK. s. (1967) Nnl,~e (I,o,rtJo,/) 215, 1491. 19. PORATH, J. (1968) i\‘ntic~ (Loixlo/O 218, 834. 20. CUATRECASAS, 1'. (1970) .I. Bir,/. Chrtn. 245, 3059. 21. SKUDE, ('7.. .4ND JEPPSSOS. J-O. (1972) Scur,~d. J. (‘/iv. Lab. Z,/rx~s/. 29 (&~p,,l 121). 55.
of Electroendosmosis-Free Agarose Exemplification of Its Use in Crossed
Gel
lmmunoelectrophoresis ANDERS
GRUBB
The method Preparation of electroendosmosis-free agarose is dcscribcd. is based upon the vovalcnt honding to agnrow of a small numhrr of poaiI irely charged groups which countcrhalance thp elec:trocnclosnlosis-llro(~~lcirl~ effect of the negatively charged groups present in commercial agarosr. It is shown how such clectroendosmosis-free agarow gel can he ~ombinrd will) advantage with polyacrylamidr pls in vrosscd ilnm~lnorlrc~tro~,hor~~i~.
Crossed immunoclectrophor~~sis can often be used with advantage instead of the classical immunoelectrophorcsis of Williams and Grabar (l), since it has a higher resolving power, it is sellliquantitativc, and it requires only a few hours (2,3). In crossed imrnunoclcctroI,horesis a first zone electrophorctic separation in an agarose gel is followed by a second electrophoresis of the separated antigens into agarose gel containing antibodies, with the electrical field perpendicular to the one used in the first zone electrophoretic separation. The resolving power of the technique is, of course, dependent on, above all, the resolving power of the first step. If the zone electrophoretic separation in agarose gel could be replaced by other common procedures for separation of macromolecules, such as polyacrylamide gel electrophoresis or isoelectric focusing in polyacrylamide gel, it would notably imrease the versatility of crossed immunoelectrophoresis. The main obstacle to replacement of the agarose gel separation by separation in polyacrylamide gel is the difference in electroendosmosis bctwecn the agarosc gel containing the antibodies and the polyacrylamide gel containing the separated ant,igens. The obstacle would be overcome if an agarose gel could be produced which, like polyacrylamidc gel, had very little or no electroendosmosis. Agar, from which agarose is refined, has long been regarded as a mixture of the two polysaccharides agarosc and agaropectin (4). The agarose has been considered a charge-free polymer and hence considered Copyright .U rights
582 @ 1973 by Academic Prrss. Inc. of reproduction in any form rwzrwd.
to form electroendosntosis-free gels when dissolved in buffers, Fvhilc agaropectin has hecn regarded a.q a negatively charged polysaccharidc and hence considered to cause the elertroendosmotic flow against the cathode in agur gels. This concept of the structurc~ of agar led to several attempts to purify the completely uncttargcd agarosc from agar (5-8~‘~ hut, although low-charged polysacchnridc~s ww ~~rodurccl, no completcl> uncharged agarosc was ohtaincd. The reason why thcsc attempts at purifying agarose wrc unsuccessful has Iwtln csplaincvl hy wwnt invcstigwtions of thv structure of agar according to nltich agar consists of a hut family of ~~olys:tccltarides with similar ni:~~rottiolcc~ulnr structures with continuously varying degrees of aciclic suhstitrtcnts and with virtually no completely unchargt~tl mol~~~tlcs (8,9 1 Thcrc thus swms to 1)~ only two ways in whic.11 clectroeticlostiiosis-fr~~~~ ngarow (‘ati tw prorluwrl. One of them is by c~lic~rrii~al rctiioval of all acitlic sut~stitucnts of thcb ha\-(5 Iwcti taken 1IO,1 1 ), agarose. Important steps in this dirwtion agarosc ,swttis to have t~ecn hut no completely elcctrocti~lost~iosis-frrc obtained. The otltcr l)ossil)lr way to l)roduw (:lcrtrocntlosmosis-free agarose is to introrlucc into tlw polysacrtiarid~~ :I .stn:tll amount of lwsit,ively charged suhstituetits which couiitcrbalan~c~ the olt~ctrortitlostiiosisI)roducing effect of tlw ncgativcly cltargcd groups I)rcacnt. in commercial agarow. This paler clcwrihw how such basic substiturttts can tw itttrodrtced into agarosc without destroying its gelling ability. Tt also demonstrates how tltc corresponding ctcrtroc~ndosmosis-fr(~(l gel can tw utilized in crossed itiiuiuttorlcrtrol~horcsis with :I iwl\-ncrylamidc gel separation in the first dimension. MATERIALS
hgarow (A-45, Ittclut~iosc, hatch 3033) was lntrchasrd from I,‘Indus-tric~ Biologiqtw Fran~aiw S.A., (knnevillicrs, Franw, Ikstrnn 40 iMT1’ 40,000, unrhargrd I from Pharmacin Fittp C’hcmirals AR, a highI> purified quality :wrylnntidr, No. 5.521S, from Hasttnan Kodak C’o.. ~V,N,~\;‘.~\~‘,-tctr:ttitcth~lct~iyteii~~~li:ttiiiti~~ :Illd .~,.\-‘-mc~tltplencl~isa~rytamide from BL)H C’ltcwicals I,tcl. ~\~..~\r’-t~~cthylcIlchis:~~ryla~~~i(le was wcrystallized once from chloroform. Atnpltolytc~ solutions wew bought from I,KB Products and cyanogen hromitle from Fluka 4.G. All other chemicals except the 1primary qua&nary diamine described hctow, ww of analytical grad(J obtained from commercial sourws. Preparation. oj i.%aminoeth~yl~t~iNlethylanlvnonilrnl bromide h!ydrobromide. 1 equivalent of anhydrous trimethylamine was conducted into nitrobenzenc containing 1 equivalent 1,2-dihromoethane during continuous stirring at room temperature. The resulting (2-hromorthyl) trimethylammonium bromide was then extracted into distilled water, which was
584
ANDERS
GRUBB
subsequently evaporated. The salt was t,hcn recrystallized from ethanol and dissolved in 10 equivalents of concentrated aqueous ammonia. After 6 days at room temperature the solution was taken to dryness by evaporation. Small amounts of yellow-colored byproducts were dissolved in a small volume of hot ethanol. The product obtained was a white cryst,alline powder. The purity of the product was tested by thin-layer chromatography on aluminium oxide coated and on silica gel coated plates (developed with n-butanol/glacial acetic acid/water, 34/34/132, v/v) as well as by high-voltage leaper-clcctrophorcsis in a phosphate buffer of pH 12.5. Staining with either ninhydrin or dipicrylamin (12) revealed only one spot in the chromatograms and in the electropherogram. During the latt’er part of this work, the intermediary product (2-bromoethyl) trimethylammonium bromide became available from Schuchardt A.G. METHODS
Introduction of positizlely charged gwups into agarose molecules. Agarose was dissolvtd to a concentration of 0.337% in boiling distilled water, and the solution was allowed to cool to room temperature. A viscous solution was formed. The pH of the solution was adjusted to 11.0 with 0.2 M sodium hydroxide. Immediately thereafter, a freshly prepared water solution of cyanogen bromide wa#s added to the agarose solution during vigorous stirring. The stirring was continued for the next 10 min, and the pH was kept, at 11.0 by small additions of the sodium hydroxide solution and the temperature at about 20°C by the addition of crushed ice. Immediately after this period, a 2.0 M solution of (2aminoethyl) trimcthylammonium bromide in water adjusted to pH 10.0 was added. The mixture was then left, at, 4°C overnight. It was then centrifuged for 15 min at lO,OOOg, which resulted in the formation of a gel at the bottom of the centrifuge tube. The supernatant was discarded, the gel shaken with dist.illed water and then centrifuged down again. The washing procedure was performed three times. The washed gel was then frozen and thercaftcr allowed t’o t’haw in a nylon net, whereby the gel lost at least 90% of its water. Finally, t’he gel was freeze-dried. Determination of electroendosmosis. The freeze-dried agarose derivative was dissolved to a concentration of 17% in a buffer solution’ on a boiling water bath, and then a 1-mm-t’hick gel was cast on a microscope ‘To avoid alkaline hydrolysis of the agarose derivative when a gel was to be prepared in a glycine buffer of pH 10.3, the derivative was dissolved in distilled water. the solution cooled to about 50°C. and then a concentrated buffer WBS added immedistelqbefore the gel was cast.
slide. This was then J~lacctl in a water-cooled elcctrophoresis apparatus with a tightly fitting lid (13) and connected with the electrode vessels by means of gel bridges cast, with the same gel as the one on the slide. A slit was filled with a solution of d&ran 40 itt the buffer used for electrophoresis, and a pot&in1 graclic~nt of I5 V;/cm was applied for 3 hr. Thereafter the slide was immersed in a (Ir,r;trntt-1)rr~~i])itatittg solution of acet.ic acid, cthsttol. and water (,5/70/‘25, Y, Y I (14). The vlect,roendosmosis was then cnlculatetl from tlicl clktattce tr:tvcIet] by the uncharged dextran, the electro])ltorcGs t.imc, :LINI the J)0t8ential gradient. Isoelectric Joc~~Gq in pol~yncryltrvride qel. The ])roccdurc described by .Jeppsson and Rergluntl (1.5,) K:~S uac~l. Polyacrylnvlitle slab elect?,ophclresis. This was performed ]y preparing a l.O-mm-thick 5:/r J’ol~--:trrylnltticl(~ gel on Ixtrt of a glass-plate and the suhsquettt casting of an cl(,~trocllc]o~;moai8-free agnroac gel on the free part of the glass-plate. ~~l)l)lic~:ttiott slits were ]~o(lu~d in the ngarose gel by inserting a mold nith 15 rc~ctnngular teeth in the agarose solution and removing it. nftt>r the, Iattcr hart sctt. The two types of gels were prepared using thv same barbital ])uffcr 1’0.07531, pH 8.6 1, and the electro])ltorcsis was perfortticd horizotttally at 20 FTi’ct~t in a simple water-cooled elcct,ropltorcsis ap]):tratus (13 i The gchl5were connected with the rlcctrotle vessels containing the lrur],ital brtfi’cr by tneans of thick electroendosmosis-frc~c~:tg:tro~c~go] IJritlgW;. This simple technique for polyacrylamitlc slab electrophorc~ris iLq (lcsc~rihcclin clotails elsewhere (16).
When increasing nmount~ of cyattogcln bronti~l~~wcrc used in the introtluction of positively chargccl grottl~ ittto :q:trose. thcx c,lcc,trocndosmclsi::
of the resulting gels was influenced in the way depicted in Fig. 1. The final concentration of (2-aminoethyl) trimethylammonium bromide was kept at 0.06 M in these experiments. The electroendosmosis was tested in 1% gels in either barbital buffer (pH 8.6; 0.075 MI or glycine buffer (pH 10.3; 0.06 M I, il’o significant difference in electroentlosmosis was found between t,he gels in the two different buffers. It can be seen that the cathodal elcctroenclosmosis of unaltered agarose diminished and was converted to an anodal one as the amount of cyanogcn bromide increased. The anodal elcctrocndosmosis increased with the amount of cyanogen bromide added until an agarosc product insoluble in boiling dist,illed water was obtained by an addit’ion of about 1.5 g cyanogen bromide/g agarose. When a fixed amount of cyanogen bromide (0.3 g/g agarosel was used to introduce positively charged groups into agarose but the final concentration of (2-aminoethyl) trirliethylarllrnonium bromide was varied, the electroendosmosis of the derived gels varied as shown in Fig. 2. The electroendosmosis was tested in l$Z gels in the two buffers mentioned above. Gels prepared in the two buffers had the same clectroendosmosis. An increase in the concentration of (2-aminoethyl) trimethylammoniumbromide caused t,he cathodal electroendosmosis of unaltered agarosc to diminish and to convert to an anodal one. To prepare an electrocndosmosis-free agarosc gel, an agarosc clcrivative was produced that, had so many positively charged groups t,hat the corresponding gel posacssed nnodal clcctroendosmosis. This was accomplished as described in Methods with the addition of 0.3 g cyanogcn bromide/g agarose and with a final concentration of 0.15 M (2-aminoELECTROEN)OSMOSlS
(lb5cn+&‘)
+0.4 1
CNBr-ADDITION
-O.& 0
0.1
1. The electroendosmosis increasing amounts of cyanogen aminoethyl) trimethylammonium c~lectroendosmosis and negative FIG.
0.2
a3
0.4
(g/g 0.5
at pH 8.6 of gels prepared by bromide and constant amounts bromide. Positive numbers numbers cathodal ektroendosmosis.
agarose) 0.6
a reaction of agarose designate
between and (2anodal
ELECTROENDOSMOSIS
(165cm%‘)
-0.8
CONCENTRATION 0
0.1
a2
al
61)
a4
2. Tlrc clcctroendoamosis at pH 5.6 of gels prepnred by 3 wattion hrtween increasing amounts of (2-;mGnwthyl) trin~~th-lxmmoniunl~~l~~n~noni~~~~ bromide and constnni amounts of agnrose and cycmogcn hromitlc~. Positirr numhrrs dcaignnte nnodal elwt,rorndosmosis nnd nrg:ltivc nrlmhrw c3ihod:rl c~lr~troenclorn~o~i~. FIG.
ethyl 1trimcth\;l:ttntnotlittttl t)rotttide. E;l(~ctrocntlosmosiu-free agarose was obtaiwd by tnising an approl~riste volume of a solution of the agarose clerivative with nnodnl clectroetttlostllosis with a solution of commercial agnrosc (,witli cathodal electrocndostnosis) . -4nalyt,ic gel clcctrophotwia of scvcrnl human plaamns with the use of a 1% elcctrocttclostnosis-free agarosc gel in barbital buffer (0.075 M ; pH 8.6) produced the protein ltattcrn s in Fig. 3A. No protein adsorption was observed dcspitc t’he prcscncc of a small number of positively charged groups in the gel. If ltuman plasma containing haptoglobin of type 2-l or 2-2 is subjccted to Itolyacrylatnide slat) elcctrophoresis, tltc polymers of the haptoglobin types will separate mainly according to their molecular size. If the lwlpmers arc allowed to tnigrate ink0 elcctroetidost~~osis-free agarose containing anti-ltal~toglot~iti serum in crossed irnmunoelectro1)horesis. tltc individual polymers will be viaualizcd as alto~~n in Figs. 4 and 5. It Call h sect1 that the series of haptoglobin 2-l ltolymers contain at least 9 diffrrertt tttoleculr~ and t,hat at least I.5 different, haptoglobin 2-2 polymers exist. If ironsaturated human plasma is subjected to isoelectric focusing between pH 4 and 6 in I~olyncrylamide gel, numerous protein bantls will appear. At least 5 of thcw rcprcsettt diffcrcnt molecular forms of transfm’rill its ShOWJl by the rroswd irnnitttioclectrol,hor~~sis of Fig. 6.
FIG;. 3. Analytic gei elcctrophoresis of sis different human plasmas (or sera) with the use of (A) a 1% electrocndosmosis-free agarose gel or (B) a 1% commercial unaltered ngarosr gel in barbital buffer (0.075 M; JIH S.6). The voltage 20 V/cm was applied for 50 min, and the proteins stained with Amide Black B.
“^
590
ANDERS
GRUBB
human plasma. 7’1~ E’rc:. 6. Crossed inlmunoelectroplloresis of ken-snturatc,d tirst, elrctrophorctic step was performed by isoelectric focusing between pH 4 and 6 of plasma in polyacqlamide gr,l and the second by rlrctrophorrais in 1% cltctrorndoamosis-frer, agarosc> .~rl in barbital buffer (0.075 M: pH 8.6) containing antitixnsfrrrin senlm.
DISCUSSION
The incorporation of positively charged groups into agarose related here is based upon the reaction, previously described, between agarose and cyanogen bromide which results in an “activated” agarose that readily reacts with primary aliphatic amines with the production of water-irisolu~)lt~ agarosc products (17-20) In this study, it was found possible to obtain :I posit,ively c~hargccl agarosc clerivatiw that was soluble in water solutions and that retainrd itu gelling ability by the
592
ANDERS
GRI’BH
very acidic proteins to the gel and, second, the ionizable groups of the gel render it unsuitable as a stabilizing medium in isoelectric focusing. ACIiSOWLEDGMENT This University
investigation of Lund.
was
supported
by
n
granf-
from
Ihe
Medical
Faculty.
REFER,ENCES 1. GRABAR, P.. AND WILLIAMS, C. A. (1953) Biochim. Biophys. Acta 10, 193. 2. LAURELL, C.-B. (1965) Anal. Biochern. 10, 358. 3. GANROT. P. 0. (1972) Scctnd. J. c’lin. Lnb. Z?luest. 29 (Suppl. 124), 39. 4. ARAKI, C. (1965) in Proc. Fifth Int. Seaweed Symp. (Young, E. G.. and McLachlan, J. L., eds.), p. 3, Pergnmon Press, Oxford. 5. HJERTBN, S. (1962) B&him. Biophys. Acta 62, 445. 6. JOHANSSON. B. G., AND STEKFLO. J. (1971) Anal. Biochem. 40, 232. 7. HJERTBN, S. (1971) J. Chromutogr.61, 73. 8. DUCKWORTII, M., AXD YAPHE, ‘11:. (1971) Cnrbohyd. Res. 16, 189. 9. IZUMI, Ii. (1971) Carbohyd. Res. 17, 227. 10. PORATH, J., JANSON, J.-C., AK~ I,.%, T. (1971) J. C’h~n~tog~. 60, 167. 11. LKs. T. (1972) J. Chyomntogr. 66, 347. 12. STAHL, E.. AND SCHORN, P. J. (1967) irl Dii~~nac~hirht-C1~o~~~~togr;~l~hic~ (StallI, ‘E.. ed.), 1,. 474, Springer-Verl:lg. Berlin. 13. JOHANSSON. I:. G. (1972) fkc~u/. J. ('/in. Lab. znzwt. 29 (SUI,II~. 124), 7. 14. WIEME, R. J. (1965) in Agnr C;cll Elcctrophoresis (Wk~mc. R. J.. ed.), 1,. 99. Elsevirr. Amsterdam. 15. JWPSYOS, J.-O., AKD HERGLUKIL S. (1972) Cliu. C’hirn. dctcc 40, 153. 16. GWBB, A. (1973) Pmt. Biol. Fl~Lls 21, in prws. 17. Ax$N, R., PORATH, J.. ASI) F:RNBAC-K. S. (1967) .Vntvre (Lor~ion) 214, 1302. 18. PORATH, J., Ask. R., AND ERSBACK. s. (1967) Nnl,~e (I,o,rtJo,/) 215, 1491. 19. PORATH, J. (1968) i\‘ntic~ (Loixlo/O 218, 834. 20. CUATRECASAS, 1'. (1970) .I. Bir,/. Chrtn. 245, 3059. 21. SKUDE, ('7.. .4ND JEPPSSOS. J-O. (1972) Scur,~d. J. (‘/iv. Lab. Z,/rx~s/. 29 (&~p,,l 121). 55.