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Preparation of tritium labelled dinotrophenyl-haptens
Journal of Imlnunolo,eical Mt`lhod,~ 2 ll972) 209- 2 1 2 . ~ North-Ilolland I'ubli,,hm,v ('olnlxmy
PREPARATION OF TRITIUM LABELLED DIN ITROPHENY L-HAPTENS R. O R I O L and .I. H U E R T A * Instiler d'hnntumd~iol,,g/c, IhOlfftal H/'ou~'ai~. 96. rue Didot, 7.567.1. Paris. ('ede.v 14
Accepted 17 (')cl~ber 1972
Received I I ()ctober 1972
Trilium labelled dinitrophenyl amim~-acids (~I" high specifit` activiD were prepared and purilied b~ adsorption on a l)lAl'-cellulosc c.lumn. Tilt' retenlion in tile column yeas stron.et`r ,.~Jib lv.o l)NP-.er~mp'~ per nn)lecule lhan v, ith one and v, JlhJn tile inonosul'JslJ0.11ed derivatives tilt' adxorption ",~.a'.;In*..ttllaled by lilt' charee anti •.fizc o l the haplt`wl. "1ht` purified haptens arc read} for use, after lilt` c~ltllllll, "~Jllloul all~, furlht'r manJpulzltion.
I. I N T R O I ) L / ( " f l O N
I lighly conjugated dinilrophenyl-JDNP) proteins are good anligens and differenl DNP-haptens (['isen and Siskind. 1'-/(,4: Valentine and (.;teen. 1967: Ashman and Melzger. 1060) have been used to study the a n l i b o d y - h a p l e n reaclion. "fritiunl labelled I)NP-haptens have been purified by thin layer chromatograph.', in silica gel using n-butanol as solvenl (Kalz et al.. l q 7 0 ) , and some l)Nl'-haptens have been sucessfully labelled with iodine by Brownstone et :.l. (1066). and by Meyer el al. (1071). bu! in general, the synthesis and purification of these con> pounds is 1101 easy to perform. A simple tnelhod o f preparing tritialed l)NP-amino-acids o l h i g h specific activity and their purification is described in this paper.
* B o u r s i e r du ('olI,L'ge tie M e d e c i n e d e s I l o p i t a u \
d e Paris+
21o
R. ()P, IOI alid .I. I I [ I I R I A
2. M A I I i R I A L AND Mt-ITI IOI)S
2. 1. Rcat, rnts l-l]uoro-2,4-diilitrobenzene-3,5,31t (FI)NB311) in benzene solution (19 (7i/ nlinole}, was obtained froill tile Radiochemical ('entre Alllershatn (l']ngland). mlllJlio-acJds and Iheir lilt)lit)- aild clJ-dJilJlrt)l'lllell)'l derivatives x~eie purchased Irtitll Sigma ('heiuical ('(). (St [.outs, Me.). I"or tile synthesis t)t tile different I)NI)-alnino acids see l)()rler and Sailgei (lt)4
2. 2. 1)1:)] l;'-r('lh~losc dr<)mat< ~gruph.r Twenty gr;.iillS of I)1)52 { W h a i l i l a l l ) w e r e pitcked in a cohtllln ( 1.2 X 30 clll) alid equilibrated wilh O. I ~1 phosphate I~uffer p l l 8. S;.iillples ()l" l lnl w e r e c h r o l n a l o g r a p h e d in tile same b u f f 0 r al a f l o w r;.lle t)t (>0 ml/h alld fr;.iclJoliS of 3 nil were collecled. The abst)rbancy al 360 rim and lhe radioaclJvily were lneasured ill each Iraclion. ]'tie i>ll-subsliluled amino-acids were delecled by the nJnhydriii reaclion.
3. i~,EStJ L I S AND I)IS('LJSSION .7.1. ,~vtllhesis
~.I" tritiated ditiitrophe#trl e-amino capr,>ic acid (DNP-e A CA 311)
0.77 pmole of cold FI)NI{ was added to I ml of henzellc c o n t a i n i n g O.O5/Jmole
of I:I)NB311 ( 1 lnCi). The betizene was evaporaled hy heating the sanlple, 12 mill at IO0~'C and tile t:I)NB311 was diluted ,.,,'Jill 20/JI of dioxane. A l't)rly ill()lar excess of e-amino caproic acid ill 50/.xl o1 5'~; Na2('() 3 was added. After I h r a l room lemperature I ml of distilled water was added and the sample was chrolnalographed eli I)liAl:-celltllose. About 15~ el + the clulcd r:.idJo:lclivily came out ill the void volume of the coltmlil, t)O'/; was slightly adsorbed and at)pe:,red ill a syimrielrJcal peak which could hc idcnlit]ed :is [)NI)-~:A('.A 311. The rcmaHlil~g t),.,: of Ihe eluled radioaciivily, lllore strongly relained, came out in a singlc peak which could he idciltified as tritiated dinJlrq~hcnol ( I)NP-O1131t ). Ihc lloe lak'l, alid Ihc I)NP-()II whicll :ire the Illosl t.'tilnnloll c())llalllJn:illlS t)[" llle haptcil l)ieparalit)ils, weie hoih eliminated wJlh lhJs cdumil. [ l i e specific activity o1 Ihe puril]ed I)NI)-~-A('A31t was I ('i/ilmlole alld t)(.)~ of lhe radJoaciivjiv was specifically bt)und b)' anli-DNI ~ ailtibodies.
Pruparation o! tritium labelled dinitrolgtenrl-haptcns" c
211
m I(5.~f~I DNP-,
2
A C A 3H
1
DNP_OH3H
3H ..... 0
s.~,_o-"
°°.... t 00
i i i,**°i "*" ...... "*%,~
200 ml
Fig. I. Purificalion o f I)NP-¢:A('A 31t in the Dl'iAl'i-ccllulosc column. The clution volume, in ml, is plotted against the radioactivity (c.p.m.) of I ,ul aliquols of each fraction.
3.2. Other DNP-aminoacids In order to see if this fractionation procedure could be used to purify other DNP-haptens a series of columns were run with 0,5 mg of different amino-acids. their mono- and di-dinitrophenyl derivatives and trace amounts o f DNP-eACA 3H and DNP-OH 311. Tile elution volumes of the different haptens tested are summarized m table I. Tile reproducibility of the elution w)lumes of the haptens, based on the position of the labelled tracers, was +- 5%. All the amino-acids tested were slightly retained by tile column, their mono-DNP derivatives came out later, and the amino-acids with two DNP-groups per molecule were the more strongly adsorbed. Tile DNP-compounds of acid residues were retained more than those of neutral and non-polar residues and those adsorbed least were the DNP-compot, nds of basic residues. In the series of DNP-derivatives of non-polar residues itle order of adsorption was DNP-glycine > DNP-alanine > I)NP-valme > DNP-leucine, showing that within DNP-derivatives of the same charge, the adsorption is inversely related to tile size o f their aliphatic side chain. The elution w)lumes of p-mononitrophenol, 2,4-dinitrophenol and 2,4,(~-trinitrophenol, in tile same column, were 138, 222 and 234 ml respectively, showing that tile nitro-groups play a role in the adsorption of the hapten, and thal tile series of mononitrophenyl and trinitrophenyl amino-acids may also be purified by this method. In conclusion, tritiated haptens or'high specific activity can be obtained in a few
R. ()RIOL and I. I I L ' I I R I A
212
I able I 1 luti~m ".,olumes (in rnll of atllilloil,,.'ids ;Jnd their l)NP-deri,.ativc,; frolll the DI]AF cellulose k'O ]II II111.
Non
MOlm-DN I'
Bi-DN P
,,u b,,ti lu led
dcr r.,;llivc
dcri;ali,.v
("?, 4temc Lysmc
26
..\rginmc
/\luilaoacid
31
44 54
lh-olme
31
67
Leucine Valine Scrim.' c-A(A
45 45
72 76 77 82 85
Melhic, nin¢ Ahlrdn0
45 36
l'hreonine
25~ 276
9I 93
(;1~, cinc PheiLvlalanme Asll~nutic acid
29
I I1~ I10
34
( ; h l l ; m l i c acM ('ysleic acid
38
158 166
'I '. rosir.,.'
45
21)1) 227
347
hours, ready for use. in c o n d i t i o n s o f p t l and ionic strength suitable for equ, i l i b r i u m studies with antibodies,
REFI!RI'~N('I!S
Ashman, R.I". and R.I'.II. Met,'ger, 1969. J. Biol. Chcnl. 244, 3405. I;Irov,,nsl,ane, A., N.A. Mitchison and R. Pill Rivers, 1966, Immunol. IO, 465. Eisen. I I N . and (,.(,. Siskind, 1964, Biochemislry 3,997. Katz. I).1t., W.I.I. Paul, I . A . Gaidl and 13. Benacerraf, 1970, .I. I'xptl. Med. 132,261. Meyer, M., M.A. l')elius, R. Mitchison, R. Pill Rivers and R. Rinde, 1971, I.~uropean J. Imm u n o l . I, 267. l'orlt.'r, R.R. and I'. San,eer, 1948, Bh~chem..I. 42. 287. Porter, RR., 1950, Methods Med. Rcs. 3,256. V,denlme. R.('. and N M . (,recn. 1967..I. Mol. Biol. 27. 615.
PREPARATION OF TRITIUM LABELLED DIN ITROPHENY L-HAPTENS R. O R I O L and .I. H U E R T A * Instiler d'hnntumd~iol,,g/c, IhOlfftal H/'ou~'ai~. 96. rue Didot, 7.567.1. Paris. ('ede.v 14
Accepted 17 (')cl~ber 1972
Received I I ()ctober 1972
Trilium labelled dinitrophenyl amim~-acids (~I" high specifit` activiD were prepared and purilied b~ adsorption on a l)lAl'-cellulosc c.lumn. Tilt' retenlion in tile column yeas stron.et`r ,.~Jib lv.o l)NP-.er~mp'~ per nn)lecule lhan v, ith one and v, JlhJn tile inonosul'JslJ0.11ed derivatives tilt' adxorption ",~.a'.;In*..ttllaled by lilt' charee anti •.fizc o l the haplt`wl. "1ht` purified haptens arc read} for use, after lilt` c~ltllllll, "~Jllloul all~, furlht'r manJpulzltion.
I. I N T R O I ) L / ( " f l O N
I lighly conjugated dinilrophenyl-JDNP) proteins are good anligens and differenl DNP-haptens (['isen and Siskind. 1'-/(,4: Valentine and (.;teen. 1967: Ashman and Melzger. 1060) have been used to study the a n l i b o d y - h a p l e n reaclion. "fritiunl labelled I)NP-haptens have been purified by thin layer chromatograph.', in silica gel using n-butanol as solvenl (Kalz et al.. l q 7 0 ) , and some l)Nl'-haptens have been sucessfully labelled with iodine by Brownstone et :.l. (1066). and by Meyer el al. (1071). bu! in general, the synthesis and purification of these con> pounds is 1101 easy to perform. A simple tnelhod o f preparing tritialed l)NP-amino-acids o l h i g h specific activity and their purification is described in this paper.
* B o u r s i e r du ('olI,L'ge tie M e d e c i n e d e s I l o p i t a u \
d e Paris+
21o
R. ()P, IOI alid .I. I I [ I I R I A
2. M A I I i R I A L AND Mt-ITI IOI)S
2. 1. Rcat, rnts l-l]uoro-2,4-diilitrobenzene-3,5,31t (FI)NB311) in benzene solution (19 (7i/ nlinole}, was obtained froill tile Radiochemical ('entre Alllershatn (l']ngland). mlllJlio-acJds and Iheir lilt)lit)- aild clJ-dJilJlrt)l'lllell)'l derivatives x~eie purchased Irtitll Sigma ('heiuical ('(). (St [.outs, Me.). I"or tile synthesis t)t tile different I)NI)-alnino acids see l)()rler and Sailgei (lt)4
2. 2. 1)1:)] l;'-r('lh~losc dr<)mat< ~gruph.r Twenty gr;.iillS of I)1)52 { W h a i l i l a l l ) w e r e pitcked in a cohtllln ( 1.2 X 30 clll) alid equilibrated wilh O. I ~1 phosphate I~uffer p l l 8. S;.iillples ()l" l lnl w e r e c h r o l n a l o g r a p h e d in tile same b u f f 0 r al a f l o w r;.lle t)t (>0 ml/h alld fr;.iclJoliS of 3 nil were collecled. The abst)rbancy al 360 rim and lhe radioaclJvily were lneasured ill each Iraclion. ]'tie i>ll-subsliluled amino-acids were delecled by the nJnhydriii reaclion.
3. i~,EStJ L I S AND I)IS('LJSSION .7.1. ,~vtllhesis
~.I" tritiated ditiitrophe#trl e-amino capr,>ic acid (DNP-e A CA 311)
0.77 pmole of cold FI)NI{ was added to I ml of henzellc c o n t a i n i n g O.O5/Jmole
of I:I)NB311 ( 1 lnCi). The betizene was evaporaled hy heating the sanlple, 12 mill at IO0~'C and tile t:I)NB311 was diluted ,.,,'Jill 20/JI of dioxane. A l't)rly ill()lar excess of e-amino caproic acid ill 50/.xl o1 5'~; Na2('() 3 was added. After I h r a l room lemperature I ml of distilled water was added and the sample was chrolnalographed eli I)liAl:-celltllose. About 15~ el + the clulcd r:.idJo:lclivily came out ill the void volume of the coltmlil, t)O'/; was slightly adsorbed and at)pe:,red ill a syimrielrJcal peak which could hc idcnlit]ed :is [)NI)-~:A('.A 311. The rcmaHlil~g t),.,: of Ihe eluled radioaciivily, lllore strongly relained, came out in a singlc peak which could he idciltified as tritiated dinJlrq~hcnol ( I)NP-O1131t ). Ihc lloe lak'l, alid Ihc I)NP-()II whicll :ire the Illosl t.'tilnnloll c())llalllJn:illlS t)[" llle haptcil l)ieparalit)ils, weie hoih eliminated wJlh lhJs cdumil. [ l i e specific activity o1 Ihe puril]ed I)NI)-~-A('A31t was I ('i/ilmlole alld t)(.)~ of lhe radJoaciivjiv was specifically bt)und b)' anli-DNI ~ ailtibodies.
Pruparation o! tritium labelled dinitrolgtenrl-haptcns" c
211
m I(5.~f~I DNP-,
2
A C A 3H
1
DNP_OH3H
3H ..... 0
s.~,_o-"
°°.... t 00
i i i,**°i "*" ...... "*%,~
200 ml
Fig. I. Purificalion o f I)NP-¢:A('A 31t in the Dl'iAl'i-ccllulosc column. The clution volume, in ml, is plotted against the radioactivity (c.p.m.) of I ,ul aliquols of each fraction.
3.2. Other DNP-aminoacids In order to see if this fractionation procedure could be used to purify other DNP-haptens a series of columns were run with 0,5 mg of different amino-acids. their mono- and di-dinitrophenyl derivatives and trace amounts o f DNP-eACA 3H and DNP-OH 311. Tile elution volumes of the different haptens tested are summarized m table I. Tile reproducibility of the elution w)lumes of the haptens, based on the position of the labelled tracers, was +- 5%. All the amino-acids tested were slightly retained by tile column, their mono-DNP derivatives came out later, and the amino-acids with two DNP-groups per molecule were the more strongly adsorbed. Tile DNP-compounds of acid residues were retained more than those of neutral and non-polar residues and those adsorbed least were the DNP-compot, nds of basic residues. In the series of DNP-derivatives of non-polar residues itle order of adsorption was DNP-glycine > DNP-alanine > I)NP-valme > DNP-leucine, showing that within DNP-derivatives of the same charge, the adsorption is inversely related to tile size o f their aliphatic side chain. The elution w)lumes of p-mononitrophenol, 2,4-dinitrophenol and 2,4,(~-trinitrophenol, in tile same column, were 138, 222 and 234 ml respectively, showing that tile nitro-groups play a role in the adsorption of the hapten, and thal tile series of mononitrophenyl and trinitrophenyl amino-acids may also be purified by this method. In conclusion, tritiated haptens or'high specific activity can be obtained in a few
R. ()RIOL and I. I I L ' I I R I A
212
I able I 1 luti~m ".,olumes (in rnll of atllilloil,,.'ids ;Jnd their l)NP-deri,.ativc,; frolll the DI]AF cellulose k'O ]II II111.
Non
MOlm-DN I'
Bi-DN P
,,u b,,ti lu led
dcr r.,;llivc
dcri;ali,.v
("?, 4temc Lysmc
26
..\rginmc
/\luilaoacid
31
44 54
lh-olme
31
67
Leucine Valine Scrim.' c-A(A
45 45
72 76 77 82 85
Melhic, nin¢ Ahlrdn0
45 36
l'hreonine
25~ 276
9I 93
(;1~, cinc PheiLvlalanme Asll~nutic acid
29
I I1~ I10
34
( ; h l l ; m l i c acM ('ysleic acid
38
158 166
'I '. rosir.,.'
45
21)1) 227
347
hours, ready for use. in c o n d i t i o n s o f p t l and ionic strength suitable for equ, i l i b r i u m studies with antibodies,
REFI!RI'~N('I!S
Ashman, R.I". and R.I'.II. Met,'ger, 1969. J. Biol. Chcnl. 244, 3405. I;Irov,,nsl,ane, A., N.A. Mitchison and R. Pill Rivers, 1966, Immunol. IO, 465. Eisen. I I N . and (,.(,. Siskind, 1964, Biochemislry 3,997. Katz. I).1t., W.I.I. Paul, I . A . Gaidl and 13. Benacerraf, 1970, .I. I'xptl. Med. 132,261. Meyer, M., M.A. l')elius, R. Mitchison, R. Pill Rivers and R. Rinde, 1971, I.~uropean J. Imm u n o l . I, 267. l'orlt.'r, R.R. and I'. San,eer, 1948, Bh~chem..I. 42. 287. Porter, RR., 1950, Methods Med. Rcs. 3,256. V,denlme. R.('. and N M . (,recn. 1967..I. Mol. Biol. 27. 615.