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Stereospecificity of murine myeloma protein-315 to enantiomeric spin labeled dinitrophenyl hapten
lmmunocheraistry, 1974, Vol. 11, pp. 377-379. Pergamon Press. Printed in Great Britain
STEREOSPECIFICITY OF M U R I N E MYELOMA PROTEIN-315 TO ENANTIOMERIC SPIN LABELED DINITROPHENYL HAPTEN* L. T. L. WONG,* L . H . PIETTE,I" J. R. LITI'LE:~ and J. C. HSIA* * Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8, t"Department of Biochemistry and Biophysics, School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, U.S.A. and :~Department of Medicine, The Jewish Hospital of St. Louis and the Departments of Medicine and Microbiology, Washington University School of Medicine, St. Louis, Missouri, U.S.A. (Received 1 November 1973)
Abstract--Binding of the unresolved enantiomers of N-(1-oxyl-2,2,5,5-tetramethyl-3-d,l-amino-pyrrolidinyl)-2,4-dinltrobenzene [DNP-SL (5)] at the polynitrophenyl binding site of a homogeneous murine myeloma protein 315 (protein-315) results in two sets of strongly immobilized resonance peaks. Resolution of the starting 1-oxyl-2,2,5,5-tetramethyl-3-d, l-amino-pyrrolidine with an optically active acid, benzoylL-leucine resulted in partial separation of the enantiomeric amines. DNP-SL (5) ligands were prepared from these enantiomers. The enantiomeric DNP-SL (5) when bound to separate aliquot of protein-315 gave rise to only one set of immobilized resonance peaks each corresponded to one of the two sets of resonance peaks of the mixed enantiomeric DNP-SL (5). Binding studies indicated that the two enantiomeric forms of DNP-SL (5) have identical binding affinity to protein-315. The effects of motional restriction and asymmetric polarity of the protein-315 binding site on the two enantiomers were discussed in terms of the rigidity of the site and the orientation of the hapten.
INTRODUC~ON
Application of nitroxide spin label as a conformational probe has contributed considerably to our understanding oftbe longitudinal dimension of the antibody hapten combining site (Hsia and Piette, 1969a; Hsia and Little, 1973), specificity (Hsia and Piette, 1969b; Hsia et al. 1973a) and change in hapten combining site during the immune response (Hsia and Little, 1971). The use ofenantiomeric spin labels as conformational probes to study antibody active site structure was first introduced by Hsia and Piette (1968; Hsia, 1968). Recently it was reported that binding of N-(1-oxyl2,2,5,5-tetramethyl-3-d,1-methylamino-pyrrolidinyl)-2, 4-dinitrobenzene (DNP-methylene-SL(5)) to murine myeloma protein-315 results in the appearance of two
02N'~NO2~NjH
sets of immobilized resonance peaks (Hsia and Little, 1973; Piette et al., 1971). Since protein-315 is known to be homogeneous and the DNP-methylene-SL(5) analytically pure, the only possible explanation was the presence of the two enantiomeric forms of DNPmethylene-SL(5). In the present study, we report that binding of mixed enantiomers of N-(l-oxyl-2,2,5,5-tetramethyl-3-d,l-amino-pyrrolidinyl)-2,4-dinitrobenzene (DNP-SL(5)) to protein-315 also gives rise to two sets of immobilized resonance peaks. We also show that DNP-SL(5) prepared from partially resolved enantiomers of 1oxyl-2,2,5,5-tetramethyl-3-d,l-amino-pyrrolidinewhen bound to protein-315 gives orily one set of immobilized resonance peaks, each corresponding to one of the two enantiomeric DNP-SL(5). These results are discussed
H'N'~O2N~"/N0202 N....~ /
o o d,t-DNP-methylene-SL (5) * Address reprint requests to: J. C. Hsia, Department of Pharmacology, University of Toronto, Toronto 181, Canada. 377
NO2
~..~/L'-..N/H
02 N~.-NO2 H"--N/",~//
o d, t-DNP-SL (5l
378
L.T.L. WONG, L. H. PIETTE, J. R. LITTLE and J. C. HSIA
in terms of the effect of motional restriction and assymetric distribution of polar and apolar groups in the ligand binding site of protein-315.
o.b
i
MATERIALS AND METHODS
Immunospecificallypurified A-myelomaprotein (protein315) produced by the mouse plasmacytoma MOPC-315 was obtained from the pooled sera of Balb/C mice according to the procedure of Eisen et al. (1968). N-(1-oxyl-2,2,5,5-tetramethyl-3-methylaminopyrrolidinyl)-2,4-dinitrobenzeneand
i
j:
J
1 I
N-(1-oxyl-2,2,5,5-tetramethyl-3-aminopyrrolidinyl)-2,4-dini-
trobenzene were prepared as previously described (l-Isiaand Piette, 1969a, 1969b). Enantiomeric 1-oxyl-2,2,5,5-tetramethyl-3-d,l-aminopyrrolidine were resolved by converting to salt derivatives with benzoyl-L-leucine (Williams and Young, 1963)and recrystallization of the salts with chloroform--ether to give a solid rich in one diastereomer and the mother liquor rich in another diastereomer; the enantiomers were regenerated from these diastereomers with triethylamine and reacted with fluorodinitrobenzene (FDNB) (Hsia and Piette, 1969b) to give the corresponding DNP spin labeled ligands. All ESR spectra were recorded on a Varian E-6 X-band ESR spectrometer at room temperature. The bindings were performed with immunoglobulin concentrations in bufferedsaline,pH 7.4,of approximately 5 x 10- 5M and spin labeled ligand concentration of approximately 1 1 0 - S ~ 2 x 10-SM.
,
I I I I I I
II
x
RESULTS
lOG
The resonance spectra of protein-315, bound and free DNP-SL(5) are shown in Fig. 1A. Two sets of broad resonance peaks a and b are well resolved due to the immobilization of the spin labeled ligand at the immuglobulin binding site; the separation between the peaks in gauss is tabulated in Table 1. Since protein-315 has been shown to be homogeneous, the two sets of strongly immobilized resonance peaks could be due to the presence of equal concentration of the enantiomeric forms of the spin label which may result in a different extent of immobilization or a different assymetric polarity environment in the combining site. To resolve these possibilities, we have resolved the enantiomeric forms of the SL(5) with the use of benzoyl-L-leucine. The partially purified amine SL(5) compounds were then H:,N H
0~'
""O"
d, t-amine SL(5)
reacted with FDNB to yield the respective enantiomeric form of the DNP ligands. The resonance spectra of the cnantiomeric DNP-SL(5) are shown in Fig. 1B and 1C. It can be seen that DNP-SL(5) prepared from the enantiomeric amine precipitated with benzoyl-L-leucine [DNP-SL(5)I] gives rise to the outer set of resonance peaks while the DNP-SL(5) prepared from the mother liquor [DNP-SL(5)II] gives rise to the inner set of
.
Fig. 1. Electron spin resonance spectra of spin labeled DNP ligands in the presence of myeloma protein-315. (1A) DNP-SL(5). (IB) DNP-SL(5)I. (1C) DNP-SL(5)II. resonance peaks. The A~= value are 62.4+0.5 g and 51.5 _ 0.5 g respectively. The different Am~ values could result from a difference in the mobilities of the enantiomeric forms of the spin label in the active site or iftbe extent of immobilization were the same, it could indicate differences in the distribution of polar and apolar residues in the combining site. Two kinds of evidence support the latter possibility. First, one would expect that DNP-methylene-SL(5) which has an extra methylene group would be a more flexible and extended molecule than DNPSL(5) and should have smaller Am~, if the difference between the enantiomeric DNP-SL(5) is due to differences in mobility of the bound ligands. However, the results show that the Am,x for DNP-methylene-SL(5) Table 1. Am~ value of the protein-315 immobilized resonance peaks Amx, G Hapten DNP-SL (5) DNP-SL (5)I DNP-SL(5)II DNP-methylene-SL{5)
a 62.4 + 0.5 62-4_+0.5 -66.3_+0.5
b 52 + 0-5 -51-5+0.5 57.2_+0.5
Use of Enantiomeric Spin Labels as Conformational Probes is greater than the Araaxfor DNP-SL(5) (Hsia and Little, 1973) (see Table 1). Therefore, the difference between the two enantiomeric forms of DNP-SL(5)omay indeed reflect an assymetric distribution of polar and apolar residues in the combining site. A second argument in support of this conclusion is derived from the binding studies of unresolved DNPSL(5) to protein-315 (Hsia et al., 1973b). These data indicate that each immunoglobulinmolecule bears two ligand binding sites constituting a population with uniform intrinsic affinity for the mixture of the two enantiomers of DNP-SL (5). DISCUSSION The change in Am~ value of antibody-bound spin labeled haptens has been used by Hsia and Piette (1969a), Hsia and Little (1971) to detect small variations in the mobility of the spin label in the antibody active site. The results from the present study suggest that factors other than mobility may also affect the A n value. This is consistant with the variations in isotropic hyperfine splitting constants of nitroxide which increase with solvent polarity (Hamilton and McConnell, 1968). Therefore, a small change in the orientation of the nitroxide ring with respect to a fixed polar residue in the combining site may also result in a change of the Am~x value such as in the case of the enantiomeric forms of DNP-SL(5). This conclusion would suggest that when hapten is bound, the protein-315 polynitrophenyl binding site exists in a fairly rigid conformation and the hapten has a fixed orientation with respect to the combining site. Failing to meet these stringent conditions, we would not expect to detect the polarity effect on the enantiomeric DNP-SL(5).
379
Acknowledgements--Supported in part by Research Grant MA-4129 and MRC-DA6 from the Medical Research Council of Canada (L.T.L.W., J.C.H.) and Research Grant AI 09723 from the National Institute of Allergy and Infectious Disease, National Institutes of Health and Research Grant T-560 from the American Cancer Society Inc. and by a Public Health Service Research Career Program award (1-K3-DM-38, 620) from the National Institute of Arthritis and Metabolic Diseases (J.R.L).
REFERENCES
Eisen M. M., Simms E. S. and Potter M. (1968) Biochemistry 7, 4126. Hamilton C. L. and McConnell H. M. (1968)Structural Chemistry and Molecular Biology (Edited by Rich A. and Davidson N.), p. 115. Freeman, San Francisco, Calif. Hsia J. C. (1968) Ph.D. Thesis, University of Hawaii. Hsia J. C. and Little J. R. (1971) Biochemistry 10, 3742. Hsia J. C. and Little J. R. (1973) FEBS Letters 31, 80. Hsia J. C. and Piette L. H. (1968) Recent Development in Magnetic Resonance in Biological System (Edited by Fujiwara S. and Piette L. H.), p. 74. Hirokawa, Tokyo. Hsia J. C. and Piette L. H. (1969a) Archs Biochem. Biophys. 129, 296. Hsia J. C. and Piette L. H. (1969b) Archs Biochem. Biophys. 132, 466. Hsia J. C., Wong L. T. L., Pryse K. and Little J. R. (1973a) lmmunochemistry 10, 517. Hsia J. C., Wong L. T. L. and Kalow W. (1973b) J. lmmun. Meth. 3, 17. Piette L. H., Hsia J. C., Kosman D. J. and Spallholz, J. E. (1971) Abstract: First European Biophysics congress (Edited by Broda E., Locker A. and Spinter-Lederer H.), p. 113. Williams M. and Young G. T. (1963) J. chem. Soc. 881.
STEREOSPECIFICITY OF M U R I N E MYELOMA PROTEIN-315 TO ENANTIOMERIC SPIN LABELED DINITROPHENYL HAPTEN* L. T. L. WONG,* L . H . PIETTE,I" J. R. LITI'LE:~ and J. C. HSIA* * Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8, t"Department of Biochemistry and Biophysics, School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, U.S.A. and :~Department of Medicine, The Jewish Hospital of St. Louis and the Departments of Medicine and Microbiology, Washington University School of Medicine, St. Louis, Missouri, U.S.A. (Received 1 November 1973)
Abstract--Binding of the unresolved enantiomers of N-(1-oxyl-2,2,5,5-tetramethyl-3-d,l-amino-pyrrolidinyl)-2,4-dinltrobenzene [DNP-SL (5)] at the polynitrophenyl binding site of a homogeneous murine myeloma protein 315 (protein-315) results in two sets of strongly immobilized resonance peaks. Resolution of the starting 1-oxyl-2,2,5,5-tetramethyl-3-d, l-amino-pyrrolidine with an optically active acid, benzoylL-leucine resulted in partial separation of the enantiomeric amines. DNP-SL (5) ligands were prepared from these enantiomers. The enantiomeric DNP-SL (5) when bound to separate aliquot of protein-315 gave rise to only one set of immobilized resonance peaks each corresponded to one of the two sets of resonance peaks of the mixed enantiomeric DNP-SL (5). Binding studies indicated that the two enantiomeric forms of DNP-SL (5) have identical binding affinity to protein-315. The effects of motional restriction and asymmetric polarity of the protein-315 binding site on the two enantiomers were discussed in terms of the rigidity of the site and the orientation of the hapten.
INTRODUC~ON
Application of nitroxide spin label as a conformational probe has contributed considerably to our understanding oftbe longitudinal dimension of the antibody hapten combining site (Hsia and Piette, 1969a; Hsia and Little, 1973), specificity (Hsia and Piette, 1969b; Hsia et al. 1973a) and change in hapten combining site during the immune response (Hsia and Little, 1971). The use ofenantiomeric spin labels as conformational probes to study antibody active site structure was first introduced by Hsia and Piette (1968; Hsia, 1968). Recently it was reported that binding of N-(1-oxyl2,2,5,5-tetramethyl-3-d,1-methylamino-pyrrolidinyl)-2, 4-dinitrobenzene (DNP-methylene-SL(5)) to murine myeloma protein-315 results in the appearance of two
02N'~NO2~NjH
sets of immobilized resonance peaks (Hsia and Little, 1973; Piette et al., 1971). Since protein-315 is known to be homogeneous and the DNP-methylene-SL(5) analytically pure, the only possible explanation was the presence of the two enantiomeric forms of DNPmethylene-SL(5). In the present study, we report that binding of mixed enantiomers of N-(l-oxyl-2,2,5,5-tetramethyl-3-d,l-amino-pyrrolidinyl)-2,4-dinitrobenzene (DNP-SL(5)) to protein-315 also gives rise to two sets of immobilized resonance peaks. We also show that DNP-SL(5) prepared from partially resolved enantiomers of 1oxyl-2,2,5,5-tetramethyl-3-d,l-amino-pyrrolidinewhen bound to protein-315 gives orily one set of immobilized resonance peaks, each corresponding to one of the two enantiomeric DNP-SL(5). These results are discussed
H'N'~O2N~"/N0202 N....~ /
o o d,t-DNP-methylene-SL (5) * Address reprint requests to: J. C. Hsia, Department of Pharmacology, University of Toronto, Toronto 181, Canada. 377
NO2
~..~/L'-..N/H
02 N~.-NO2 H"--N/",~//
o d, t-DNP-SL (5l
378
L.T.L. WONG, L. H. PIETTE, J. R. LITTLE and J. C. HSIA
in terms of the effect of motional restriction and assymetric distribution of polar and apolar groups in the ligand binding site of protein-315.
o.b
i
MATERIALS AND METHODS
Immunospecificallypurified A-myelomaprotein (protein315) produced by the mouse plasmacytoma MOPC-315 was obtained from the pooled sera of Balb/C mice according to the procedure of Eisen et al. (1968). N-(1-oxyl-2,2,5,5-tetramethyl-3-methylaminopyrrolidinyl)-2,4-dinitrobenzeneand
i
j:
J
1 I
N-(1-oxyl-2,2,5,5-tetramethyl-3-aminopyrrolidinyl)-2,4-dini-
trobenzene were prepared as previously described (l-Isiaand Piette, 1969a, 1969b). Enantiomeric 1-oxyl-2,2,5,5-tetramethyl-3-d,l-aminopyrrolidine were resolved by converting to salt derivatives with benzoyl-L-leucine (Williams and Young, 1963)and recrystallization of the salts with chloroform--ether to give a solid rich in one diastereomer and the mother liquor rich in another diastereomer; the enantiomers were regenerated from these diastereomers with triethylamine and reacted with fluorodinitrobenzene (FDNB) (Hsia and Piette, 1969b) to give the corresponding DNP spin labeled ligands. All ESR spectra were recorded on a Varian E-6 X-band ESR spectrometer at room temperature. The bindings were performed with immunoglobulin concentrations in bufferedsaline,pH 7.4,of approximately 5 x 10- 5M and spin labeled ligand concentration of approximately 1 1 0 - S ~ 2 x 10-SM.
,
I I I I I I
II
x
RESULTS
lOG
The resonance spectra of protein-315, bound and free DNP-SL(5) are shown in Fig. 1A. Two sets of broad resonance peaks a and b are well resolved due to the immobilization of the spin labeled ligand at the immuglobulin binding site; the separation between the peaks in gauss is tabulated in Table 1. Since protein-315 has been shown to be homogeneous, the two sets of strongly immobilized resonance peaks could be due to the presence of equal concentration of the enantiomeric forms of the spin label which may result in a different extent of immobilization or a different assymetric polarity environment in the combining site. To resolve these possibilities, we have resolved the enantiomeric forms of the SL(5) with the use of benzoyl-L-leucine. The partially purified amine SL(5) compounds were then H:,N H
0~'
""O"
d, t-amine SL(5)
reacted with FDNB to yield the respective enantiomeric form of the DNP ligands. The resonance spectra of the cnantiomeric DNP-SL(5) are shown in Fig. 1B and 1C. It can be seen that DNP-SL(5) prepared from the enantiomeric amine precipitated with benzoyl-L-leucine [DNP-SL(5)I] gives rise to the outer set of resonance peaks while the DNP-SL(5) prepared from the mother liquor [DNP-SL(5)II] gives rise to the inner set of
.
Fig. 1. Electron spin resonance spectra of spin labeled DNP ligands in the presence of myeloma protein-315. (1A) DNP-SL(5). (IB) DNP-SL(5)I. (1C) DNP-SL(5)II. resonance peaks. The A~= value are 62.4+0.5 g and 51.5 _ 0.5 g respectively. The different Am~ values could result from a difference in the mobilities of the enantiomeric forms of the spin label in the active site or iftbe extent of immobilization were the same, it could indicate differences in the distribution of polar and apolar residues in the combining site. Two kinds of evidence support the latter possibility. First, one would expect that DNP-methylene-SL(5) which has an extra methylene group would be a more flexible and extended molecule than DNPSL(5) and should have smaller Am~, if the difference between the enantiomeric DNP-SL(5) is due to differences in mobility of the bound ligands. However, the results show that the Am,x for DNP-methylene-SL(5) Table 1. Am~ value of the protein-315 immobilized resonance peaks Amx, G Hapten DNP-SL (5) DNP-SL (5)I DNP-SL(5)II DNP-methylene-SL{5)
a 62.4 + 0.5 62-4_+0.5 -66.3_+0.5
b 52 + 0-5 -51-5+0.5 57.2_+0.5
Use of Enantiomeric Spin Labels as Conformational Probes is greater than the Araaxfor DNP-SL(5) (Hsia and Little, 1973) (see Table 1). Therefore, the difference between the two enantiomeric forms of DNP-SL(5)omay indeed reflect an assymetric distribution of polar and apolar residues in the combining site. A second argument in support of this conclusion is derived from the binding studies of unresolved DNPSL(5) to protein-315 (Hsia et al., 1973b). These data indicate that each immunoglobulinmolecule bears two ligand binding sites constituting a population with uniform intrinsic affinity for the mixture of the two enantiomers of DNP-SL (5). DISCUSSION The change in Am~ value of antibody-bound spin labeled haptens has been used by Hsia and Piette (1969a), Hsia and Little (1971) to detect small variations in the mobility of the spin label in the antibody active site. The results from the present study suggest that factors other than mobility may also affect the A n value. This is consistant with the variations in isotropic hyperfine splitting constants of nitroxide which increase with solvent polarity (Hamilton and McConnell, 1968). Therefore, a small change in the orientation of the nitroxide ring with respect to a fixed polar residue in the combining site may also result in a change of the Am~x value such as in the case of the enantiomeric forms of DNP-SL(5). This conclusion would suggest that when hapten is bound, the protein-315 polynitrophenyl binding site exists in a fairly rigid conformation and the hapten has a fixed orientation with respect to the combining site. Failing to meet these stringent conditions, we would not expect to detect the polarity effect on the enantiomeric DNP-SL(5).
379
Acknowledgements--Supported in part by Research Grant MA-4129 and MRC-DA6 from the Medical Research Council of Canada (L.T.L.W., J.C.H.) and Research Grant AI 09723 from the National Institute of Allergy and Infectious Disease, National Institutes of Health and Research Grant T-560 from the American Cancer Society Inc. and by a Public Health Service Research Career Program award (1-K3-DM-38, 620) from the National Institute of Arthritis and Metabolic Diseases (J.R.L).
REFERENCES
Eisen M. M., Simms E. S. and Potter M. (1968) Biochemistry 7, 4126. Hamilton C. L. and McConnell H. M. (1968)Structural Chemistry and Molecular Biology (Edited by Rich A. and Davidson N.), p. 115. Freeman, San Francisco, Calif. Hsia J. C. (1968) Ph.D. Thesis, University of Hawaii. Hsia J. C. and Little J. R. (1971) Biochemistry 10, 3742. Hsia J. C. and Little J. R. (1973) FEBS Letters 31, 80. Hsia J. C. and Piette L. H. (1968) Recent Development in Magnetic Resonance in Biological System (Edited by Fujiwara S. and Piette L. H.), p. 74. Hirokawa, Tokyo. Hsia J. C. and Piette L. H. (1969a) Archs Biochem. Biophys. 129, 296. Hsia J. C. and Piette L. H. (1969b) Archs Biochem. Biophys. 132, 466. Hsia J. C., Wong L. T. L., Pryse K. and Little J. R. (1973a) lmmunochemistry 10, 517. Hsia J. C., Wong L. T. L. and Kalow W. (1973b) J. lmmun. Meth. 3, 17. Piette L. H., Hsia J. C., Kosman D. J. and Spallholz, J. E. (1971) Abstract: First European Biophysics congress (Edited by Broda E., Locker A. and Spinter-Lederer H.), p. 113. Williams M. and Young G. T. (1963) J. chem. Soc. 881.