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Strong evidence for the freedom of rotation of immunoglobulin G subunits
J. Mol. Biol. (1970) 50, 569-572
LETTERS TO THE EDITOR
Strong Evidence for the Freedom of Rotation of Immunoglobulin G Subunits The values of the rotational relaxation times of the intact immunoglobulin G molecule are very close to those of its papain Fab fragments. This points directly to the existence of the rotational freedom of immunoglobulin G subunits corresponding to active Fab fragments. The flexibility of bonds between subunits bearing the antibody-combining sites is an important property of antibodies belonging to this class, because it favours combination with antigens.
By measuring the amount of fluorescence polarisation of conjugatea of macromolecules with a fluorescent label, it is possible to obtain information on the form and structure of the macromolecules and, in particular, to determine whether this structure is rigid or flexible. The investigation, by this method, of conjugates of immunoglobulin G with the l-dimethylaminonaphthalene-5sulphonyl group has givenvalues for the rotational relaxation time ph of 170 to 220 nanoseconds (Wahl & Weber, 1967; W&man & Edelman, 1967). A fairly similar value of p,, was also obtained by calculation made on the assumption that the IgGt molecule might be considered as a rigid prolate ellipsoid of revolution with parameters determined by a number of physico-chemical measurements (Edehnan & Gall, 1969). Yet the conclusion about the rigidity of the IgG molecule reached on the ground of this evidence proved to be incorrect, because pa was calculated using the value of the lifetime of the excited state 7, estimated for the DNS conjugates of bovine serum albumin and of a number of other proteins, and found to be 13 nanoseconds. We have already shown that 7 for DNS-IgG is much lower, being 7.3 nanoseconds, and the values of ph for this protein calculated from fluorescence polarisation data were found to be 60 nanoseconds (Zagyansky, Nezlin & Turner-man, 1969). This result points directly to the fact that the observed degree of fluorescence polarisation is determined by Brownian rotation not of the whole IgG molecule but of its parts, which are substantially smaller and interconnected by sufficiently flexible bonds. It would be reasonable to suggest that such parts are represented by IgG subunits, corresponding to papain Fab and PC fragments. This suggestion has been partly substantiated by electron microscopy (Valentine t Green, 1967) as well as by a number of other investigation8 (Noelken, Nelson, Buckley & Tanford, 1965; Charlwood & Utsumi, 1969). Indications of some flexibility of IgG molecules were recently obtained also by measurements of the decay of the polarized fluorescence (Wahl, 1969; Fayet & Wahl, 1969). Conclusive proof of the existence of the rotational freedom of IgG subunits would be provided by establishing that the values of ph of IgG papain fragments are near the values of ph for the whole IgG molecule (Zagyansky et al., 1969). In the experiments reported below, data were obtained which convincingly prove this suggestion. Human IgG was isolated from the commercial y-globulin preparation by chromatography on DEAE-cellulose and stained according to Steiner & McAlister (1967). The 669
570
R.
S. NEZLIX,
Y. A. ZAGYANSKY
AND
L. rZ. TUMERMAN
resulting DNS-IgG conjugate was digested by papain at pH 7.5 in the presence of 0.01 M-cysteine for four hours. The papain fragments Fab and Fc were separated on a DEAE-cellulose column as described by Edelman, Heremans, Heremans & Kunkel (1960). In order to remove aggregates (and in the case of fragments the undigested IgG) before conducting polarisation measurements, the solutions of DNS conjugates were passed through a G200 Sephadex column (Zagyansky et al., 1969). The sedimentation coefficient S20,wwas found to be 6.5 s for IgG and 3.7 s for Fab and Fc. The molar ratios of DNS to protein, calculated from the absorbancy at 280 and 340 nm (accounting for protein absorbancy at 340 nm) were 3.8 for DNS-IgG, 1.55 for DNSFab, and 0.75 for DNS-Fc. The molecular weight of IgG was taken as 150,000; that of fragments as 50,000. The absorption coefficients of IgG and fragments were taken following Noelken et al. (1965) and the molar extinction of the DNS group following Chen (1968). 7 was determined on the phase fluorometer; fluorescence polarisation was estimated by means of a device with a rotating analyser (Zagyansky et al., 1969). In order to allow for the thermally activated rotation of DNS groups in DNS conjugates, measurements were carried out at a constant temperature (28°C) in solutions the viscosity of which was changed by adding different concentrations of sucrose (Weltman & Edelman, 1967; Wahl & Weber, 1967). The results of fluorescence polarisation measurements of DNS-IgG, DNS-Fab and DNS-Fc are shown in Figure 1 as isotherms, each corresponding to the change in viscosity on addition of sucrose. Extrapolation of the linear part of these isotherms to TABLE 1
Parameters of the jluorescence of DNfl-Protein DNS-protein conjugate
7 (nsec)
DNS-IgG DNS-Fab DNS-Fc
7.5 7.9 6.9
1
conjugates Ph
(nsec)
z
2.82
60
2.72
64 33
2.92
the vertical axis gave the corresponding values of l/p; (see Table 1). In Table 1 are also listed the measured values of 7. Using these values of 7 and l/plO, we have calculated, by the approximate formula of Weber (1952),
+(;-;)(l+~) P
(1)
the values of p,, at T/q = 3.34 x lo4 deg./poise (water at 25°C). The data obtained are given in Table 1. Formula (1) is valid only for the exponential fluorescence decay law. It was shown recently, however (Chen, 1968; Wahl, 1969), that the fluorescence of DNS molecules conjugated with some proteins, in particular with rabbit y-globulin, may follow the more complicated decay law. For an arbitrary fluorescence decay law @ (t), formula (1) must be replaced by the more general relation (2): t Abbreviations sulphonyl group.
used:
TgG,
immunoglobulin
G;
DNS,
1-dimethylaminonaphthalene&
LETTERS
TO
THE
EDITOR
571
UP - l/3
(2)
lb0 - 1’3 = p(t).
21 0
I 1
I 2
I 3
I 4/s
T/7x 1O-4 FIQ. 1. Dependence of reciprocal of the fluorescence polarization upon temperature visoosity for DNS-IgG, DNS-Fab and DNS-Fo. The initial buffer was 0.28 M-NaCl-0.1 M-niS-cl, pH 8.0. -@-a--, DNS-IgG protein concentration was O.OS274; --A-A-, DNS-Fab (0+087%); -O-O-, (0.061%).
established by one of us (Tumerman, 1941). One can see from equation general the fluorescence polarization depends on the exact form of the decay. But if we can replace exp( - Kt) by (1 - Kt) as in our case under we return again to formula (l), in which the decay constant T must be mean fluorescence duration t as determined by relation (3): &D (t)dt G&---F
divided
by
(the initial DNS-Fo
(2) that in fluorescence the integral replaced by
(3)
J-@VW The values of T measured on the phase fluorometer are for an arbitrary fluorescence decay law O(t) very close to f (Kuznezova, Sveshnikov & Shirokov, 1957). Thus the values of p,, given in Table 1 are correct even if fluorescence does not decay exponentially. If the values of ph for l?ab and Fc were valid not only for isolated fragments but also for independently rotating IgG subunits in situ, we could apply in our case Weber’s formula (Weber, 1952) for the mixture of molecules with different p,, values. In that
572
R. S. NEZLIN,
Y. A. ZAGYANSKY
AND
L. A. TUMERMAN
case, according to the formula, ph for the IgG molecule would be 56 nanoseconds. The value found experimentally has already been stated to be 60 nanoseconds. These data leave no doubt that the observed fluorescence polarisation of DNS-IgG is determined by Brownian rotation of subunits rather similar to Fab and Fc fragments and connected by sufficiently flexible bonds. The value of fh found experimentally for Fab is in good agreement with ph calcu-, lated for the rigid ellipsoid of revolution with 1: 2 axis ratio (Valentine & Green, 1967). The data obtained are in keeping with a number of observations that also point to the compactness of Fab and a lesser lability of its structure (Noelken et al., 1965; Abaturov Nezlin, Vengerova & Varshavsky, 1969). On the ot’her hand, the experimentally measured pi, for Fe, which is rather similar in size to Fab, is considerably lower. This points to a definite lability of the Fc structure that is also substantiated by its greater sensitivity to proteolysis and conformational changes with different pH (Charlwood & Utsumi, 1969; Abaturov et al., 1969). The independence of Brownian rotation of subunits should not affect the behaviour of the molecule as such in solution. In particular, if the linear arrangement of subunits in the IgG molecule corresponds to the minimum free energy, this molecule will behave in solution as a rod or elongated ellipsoid, as substantiated by hydrodynamic and other investigations (Edelman & Gall, 1969). On the other hand, the flexibility of bonds between subunits makes it possible for the IgG molecule to change its form under the action of comparatively weak forces such as those due to the interaction between antibody and antigen. This investigation (Immunology Unit).
received
financial
support
from the World
1969, and in revised form 14 February
Organization
R. S.NEZLIN
Institute of Molecular Biology Academy of Sciences of the U.S.S.R. Moscow, B-312, U.S.S.R. Received 29 September
Health
Y. A. ZAQYANSRY
L.A.TUMERMAN 1970
REFERENCES Abaturov, L. V., Nezlin, R. S., Vengerova, T. I. & Varshavsky, J. M. (1969). Biochim. biophya. A&, 194, 386. Charlwood, P. A. & Utsumi, S. (1969). Biochem. J. 112, 357. Chen, R. F. (1968). Arch. Biochem. Biophys. 128, 163. Edelman, G. M. & Gall, W. E. (1969). Ann. Rev. Biochem. 38, 415. Edehnan, G. M., Heremans, J. F., Heremans, M.Th. & Kunkel, H. G. (1960). J. Exp. Med. 112, 203. Fayet, M. & Wahl, P. (1969). Biochim. biophys. Acta, 181, 373. Kuznezova, L. A., Sveshnikov, B. Y. & Shirokov, V. I. (1957). Optika spectrockopia, U.S.S.R., 2, 578. Noelken, M. E., Nelson, C. A., Buckley, C. E. & Tanford, C. (1965). ,7. Biol. Chem. 240,218. Steiner, R. F. & McAlister, A. J. (1967). J. Polymer Sci. 24, 105. Tumerman, L. A. (1941). Doklady Akademii Nauk U.S.S.R. 32, 474. Valentine, E. C. & Green, N. N. (1967). J. Mol. Biol. 27, 616. Wahl, P. (1969). Biochim. biophys. Acta, 175, 55. Wahl, P. & Weber, G. (1967). J. Mol. Biol. 30, 371. Weber, G. (1952). Biochem. J. 51, 145. Weltman. J. K. & Edelman, G. M. (1967). Biochemistry, 6, 1437. Zagyansky, Y. A., Nezlin, R. S. & Tumermen, L. A. (1969). Immunochemistry, 6, 787.
LETTERS TO THE EDITOR
Strong Evidence for the Freedom of Rotation of Immunoglobulin G Subunits The values of the rotational relaxation times of the intact immunoglobulin G molecule are very close to those of its papain Fab fragments. This points directly to the existence of the rotational freedom of immunoglobulin G subunits corresponding to active Fab fragments. The flexibility of bonds between subunits bearing the antibody-combining sites is an important property of antibodies belonging to this class, because it favours combination with antigens.
By measuring the amount of fluorescence polarisation of conjugatea of macromolecules with a fluorescent label, it is possible to obtain information on the form and structure of the macromolecules and, in particular, to determine whether this structure is rigid or flexible. The investigation, by this method, of conjugates of immunoglobulin G with the l-dimethylaminonaphthalene-5sulphonyl group has givenvalues for the rotational relaxation time ph of 170 to 220 nanoseconds (Wahl & Weber, 1967; W&man & Edelman, 1967). A fairly similar value of p,, was also obtained by calculation made on the assumption that the IgGt molecule might be considered as a rigid prolate ellipsoid of revolution with parameters determined by a number of physico-chemical measurements (Edehnan & Gall, 1969). Yet the conclusion about the rigidity of the IgG molecule reached on the ground of this evidence proved to be incorrect, because pa was calculated using the value of the lifetime of the excited state 7, estimated for the DNS conjugates of bovine serum albumin and of a number of other proteins, and found to be 13 nanoseconds. We have already shown that 7 for DNS-IgG is much lower, being 7.3 nanoseconds, and the values of ph for this protein calculated from fluorescence polarisation data were found to be 60 nanoseconds (Zagyansky, Nezlin & Turner-man, 1969). This result points directly to the fact that the observed degree of fluorescence polarisation is determined by Brownian rotation not of the whole IgG molecule but of its parts, which are substantially smaller and interconnected by sufficiently flexible bonds. It would be reasonable to suggest that such parts are represented by IgG subunits, corresponding to papain Fab and PC fragments. This suggestion has been partly substantiated by electron microscopy (Valentine t Green, 1967) as well as by a number of other investigation8 (Noelken, Nelson, Buckley & Tanford, 1965; Charlwood & Utsumi, 1969). Indications of some flexibility of IgG molecules were recently obtained also by measurements of the decay of the polarized fluorescence (Wahl, 1969; Fayet & Wahl, 1969). Conclusive proof of the existence of the rotational freedom of IgG subunits would be provided by establishing that the values of ph of IgG papain fragments are near the values of ph for the whole IgG molecule (Zagyansky et al., 1969). In the experiments reported below, data were obtained which convincingly prove this suggestion. Human IgG was isolated from the commercial y-globulin preparation by chromatography on DEAE-cellulose and stained according to Steiner & McAlister (1967). The 669
570
R.
S. NEZLIX,
Y. A. ZAGYANSKY
AND
L. rZ. TUMERMAN
resulting DNS-IgG conjugate was digested by papain at pH 7.5 in the presence of 0.01 M-cysteine for four hours. The papain fragments Fab and Fc were separated on a DEAE-cellulose column as described by Edelman, Heremans, Heremans & Kunkel (1960). In order to remove aggregates (and in the case of fragments the undigested IgG) before conducting polarisation measurements, the solutions of DNS conjugates were passed through a G200 Sephadex column (Zagyansky et al., 1969). The sedimentation coefficient S20,wwas found to be 6.5 s for IgG and 3.7 s for Fab and Fc. The molar ratios of DNS to protein, calculated from the absorbancy at 280 and 340 nm (accounting for protein absorbancy at 340 nm) were 3.8 for DNS-IgG, 1.55 for DNSFab, and 0.75 for DNS-Fc. The molecular weight of IgG was taken as 150,000; that of fragments as 50,000. The absorption coefficients of IgG and fragments were taken following Noelken et al. (1965) and the molar extinction of the DNS group following Chen (1968). 7 was determined on the phase fluorometer; fluorescence polarisation was estimated by means of a device with a rotating analyser (Zagyansky et al., 1969). In order to allow for the thermally activated rotation of DNS groups in DNS conjugates, measurements were carried out at a constant temperature (28°C) in solutions the viscosity of which was changed by adding different concentrations of sucrose (Weltman & Edelman, 1967; Wahl & Weber, 1967). The results of fluorescence polarisation measurements of DNS-IgG, DNS-Fab and DNS-Fc are shown in Figure 1 as isotherms, each corresponding to the change in viscosity on addition of sucrose. Extrapolation of the linear part of these isotherms to TABLE 1
Parameters of the jluorescence of DNfl-Protein DNS-protein conjugate
7 (nsec)
DNS-IgG DNS-Fab DNS-Fc
7.5 7.9 6.9
1
conjugates Ph
(nsec)
z
2.82
60
2.72
64 33
2.92
the vertical axis gave the corresponding values of l/p; (see Table 1). In Table 1 are also listed the measured values of 7. Using these values of 7 and l/plO, we have calculated, by the approximate formula of Weber (1952),
+(;-;)(l+~) P
(1)
the values of p,, at T/q = 3.34 x lo4 deg./poise (water at 25°C). The data obtained are given in Table 1. Formula (1) is valid only for the exponential fluorescence decay law. It was shown recently, however (Chen, 1968; Wahl, 1969), that the fluorescence of DNS molecules conjugated with some proteins, in particular with rabbit y-globulin, may follow the more complicated decay law. For an arbitrary fluorescence decay law @ (t), formula (1) must be replaced by the more general relation (2): t Abbreviations sulphonyl group.
used:
TgG,
immunoglobulin
G;
DNS,
1-dimethylaminonaphthalene&
LETTERS
TO
THE
EDITOR
571
UP - l/3
(2)
lb0 - 1’3 = p(t).
21 0
I 1
I 2
I 3
I 4/s
T/7x 1O-4 FIQ. 1. Dependence of reciprocal of the fluorescence polarization upon temperature visoosity for DNS-IgG, DNS-Fab and DNS-Fo. The initial buffer was 0.28 M-NaCl-0.1 M-niS-cl, pH 8.0. -@-a--, DNS-IgG protein concentration was O.OS274; --A-A-, DNS-Fab (0+087%); -O-O-, (0.061%).
established by one of us (Tumerman, 1941). One can see from equation general the fluorescence polarization depends on the exact form of the decay. But if we can replace exp( - Kt) by (1 - Kt) as in our case under we return again to formula (l), in which the decay constant T must be mean fluorescence duration t as determined by relation (3): &D (t)dt G&---F
divided
by
(the initial DNS-Fo
(2) that in fluorescence the integral replaced by
(3)
J-@VW The values of T measured on the phase fluorometer are for an arbitrary fluorescence decay law O(t) very close to f (Kuznezova, Sveshnikov & Shirokov, 1957). Thus the values of p,, given in Table 1 are correct even if fluorescence does not decay exponentially. If the values of ph for l?ab and Fc were valid not only for isolated fragments but also for independently rotating IgG subunits in situ, we could apply in our case Weber’s formula (Weber, 1952) for the mixture of molecules with different p,, values. In that
572
R. S. NEZLIN,
Y. A. ZAGYANSKY
AND
L. A. TUMERMAN
case, according to the formula, ph for the IgG molecule would be 56 nanoseconds. The value found experimentally has already been stated to be 60 nanoseconds. These data leave no doubt that the observed fluorescence polarisation of DNS-IgG is determined by Brownian rotation of subunits rather similar to Fab and Fc fragments and connected by sufficiently flexible bonds. The value of fh found experimentally for Fab is in good agreement with ph calcu-, lated for the rigid ellipsoid of revolution with 1: 2 axis ratio (Valentine & Green, 1967). The data obtained are in keeping with a number of observations that also point to the compactness of Fab and a lesser lability of its structure (Noelken et al., 1965; Abaturov Nezlin, Vengerova & Varshavsky, 1969). On the ot’her hand, the experimentally measured pi, for Fe, which is rather similar in size to Fab, is considerably lower. This points to a definite lability of the Fc structure that is also substantiated by its greater sensitivity to proteolysis and conformational changes with different pH (Charlwood & Utsumi, 1969; Abaturov et al., 1969). The independence of Brownian rotation of subunits should not affect the behaviour of the molecule as such in solution. In particular, if the linear arrangement of subunits in the IgG molecule corresponds to the minimum free energy, this molecule will behave in solution as a rod or elongated ellipsoid, as substantiated by hydrodynamic and other investigations (Edelman & Gall, 1969). On the other hand, the flexibility of bonds between subunits makes it possible for the IgG molecule to change its form under the action of comparatively weak forces such as those due to the interaction between antibody and antigen. This investigation (Immunology Unit).
received
financial
support
from the World
1969, and in revised form 14 February
Organization
R. S.NEZLIN
Institute of Molecular Biology Academy of Sciences of the U.S.S.R. Moscow, B-312, U.S.S.R. Received 29 September
Health
Y. A. ZAQYANSRY
L.A.TUMERMAN 1970
REFERENCES Abaturov, L. V., Nezlin, R. S., Vengerova, T. I. & Varshavsky, J. M. (1969). Biochim. biophya. A&, 194, 386. Charlwood, P. A. & Utsumi, S. (1969). Biochem. J. 112, 357. Chen, R. F. (1968). Arch. Biochem. Biophys. 128, 163. Edelman, G. M. & Gall, W. E. (1969). Ann. Rev. Biochem. 38, 415. Edehnan, G. M., Heremans, J. F., Heremans, M.Th. & Kunkel, H. G. (1960). J. Exp. Med. 112, 203. Fayet, M. & Wahl, P. (1969). Biochim. biophys. Acta, 181, 373. Kuznezova, L. A., Sveshnikov, B. Y. & Shirokov, V. I. (1957). Optika spectrockopia, U.S.S.R., 2, 578. Noelken, M. E., Nelson, C. A., Buckley, C. E. & Tanford, C. (1965). ,7. Biol. Chem. 240,218. Steiner, R. F. & McAlister, A. J. (1967). J. Polymer Sci. 24, 105. Tumerman, L. A. (1941). Doklady Akademii Nauk U.S.S.R. 32, 474. Valentine, E. C. & Green, N. N. (1967). J. Mol. Biol. 27, 616. Wahl, P. (1969). Biochim. biophys. Acta, 175, 55. Wahl, P. & Weber, G. (1967). J. Mol. Biol. 30, 371. Weber, G. (1952). Biochem. J. 51, 145. Weltman. J. K. & Edelman, G. M. (1967). Biochemistry, 6, 1437. Zagyansky, Y. A., Nezlin, R. S. & Tumermen, L. A. (1969). Immunochemistry, 6, 787.