Spin-label approach to conformational properties of immunoglobulins

lmmunochemistry, 1976, Vol. 13, pp, 1001-1008. Pergamon Press. Printed in Great Britain

REVIEW ARTICLE S P I N - L A B E L A P P R O A C H TO C O N F O R M A T I O N A L

PROPERTIES OF IMMUNOGLOBULINS A. I. K,~IVAR,~INEN and R. S. NEZLIN Institute of Molecular Biology, Moscow, and Institute of Biology. Pctrozavodsk, The U.S.S.R. Academy of Sciences, U.S.S.R.

(Received 19 April 1976)

INTRODUCTION

Spin-label method developed by H. M. McConnel in 1965 is one of the most sensitive physical methods providing information on conformational properties of macromolecules. The spin-label is a stable nitroxyl radical with a general structure shown in Fig. 1. The specificity of this approach lies in providing information on local bipolymer changes in the area of label bonding. The theory and practice of this method concerning biopolymers have been extensively described (McConnel & McFarland, 1970; Smith, 1971; Jost & Griffith, 1972; Lichtenstein, 1974). This review deals with principal results obtained by this method on studying the general structure of antibodies and its changes.

CH3/CH3 O"-L'N ~,

~---"'-R

tension, and their dependence on microwave radiation strength (Kivelson, 1960; Stone et al., 1965; Kuznetsov et al., 1971 ; McCalley et al., 1972; Shimshick & McConnel, 1972; Goldman et al., 1973; Hyde & Dalton, 1972; Thomas & McConnel, 1974). The correlation time characterises the rotation velocity of N - O label group and may be qualitatively represented as a value opposite to that of the frequency of label rotation. When the label is not rigidly bonded to protein, the change in its correlation time can reflect conformational changes in label environment. I. 2. Determination of correlation time of spin-labeled proteins When the label is rigidly bonded to protein, that is its N - O group has no independent rotation freedom in respect to protein, the correlation time egtimated from ESR spectrum is approximately equal to the correlation time of macromolecule (ZM) (McCalley et al., 1972; Shimshick & McConnel, 1972). In accordance with Stokes-Einstein formula: rM = 4/3z a s ~l/T

CH3 CH3 Fig. 1. General structure of spin labels, R determines the specificity of the reaction with protein.

1. D I F F E R E N T METHOD

APPLICATIONS

OF SPIN-LABEL

FOR THE STUDY OF BIOPOLYMERS

I. 1. Determination of correlation time of spin-labels

(1)

where a is the effective Stokes radius of the macromolecule, r/is the solution viscosity, and T is the absolute temperature. With z M and rl/T known it is always possible to determine a. When the N - O group of the label, covalently bonded to protein retains its freedom of rotation, its ESR spectrum is determined by the intensity of Brownian rotation of the label relative to the macromolecule and by the velocity of rotational diffusion of the macromolecule as such. The change in the correlation time of the N - O group can therefore be caused not only by the change in the microenvironment of the label but also in the effective Stokes radius of the macromolecule. We have shown (K~iiv~ir~iinen, 1975; Nezlin et al., 1973) that if the movement of the N4D label group in respect to the macromolecule is close to the isotropic one, the following equation is valid:

ESR spectrum of spin-labels has a triplet form (Figs. 2a and b). The relation of the width of these 3 peaks is determined by averaging the anisotropy of g-factor and hyperfine interaction of nitrogen nucleus spins with unpaired electron of the N - O label group as a result of its rotation. In some cases ESR spectrum presents the overlapping of 2 spectra corresponding to two label states of different mobility (Fig. 2c). 1/TR + U = 1/'CR + 1/ZM (2) There are various techniques enabling the label correlation time to be estimated over the range of where zR + M is the resulting correlation time of the 10- t t-10- 3 sec in terms of the width relation of these N--O label group, estimated from ESR spectrum, ZR 3 peaks, their position on the axis of magnetic field is proper correlation time of the N - O spin-label 1001

1002

A.I. K)~IV)~R,Z,INEN and R. S. NEZLIN

groups, and zM is the correlation time of the macromolecule determined by equation (1). To estimate the standard value of z~a"d use can be made of the following equation: r

=

-- + "rR

stan~' Z~

.

(3)

If T/rl is changed within narrow limits (1 - 5) 10-s deg/p by varying the solution viscosity at a constant temperature, changes in zR can be neglected and the dependence of (1/TR + U)T on (T/~/)T will be represented by a straight line set at an angle 7 and intercepting the ordinate axis at the point: 1/TR+M = 1/ZR.

Thereby from equation (3): ( r / / T ) sta"d

sta.a is the correlation time of the macromolewhere zM cule corresponded to standard conditions (water, 25°C, (tilT) sta"a = 3.10 -5 P/deg). This method makes it thus possible to obtain simultaneously reliable information not only on changes in the microenvironment of the spin-label (AzR) but also the structural rearrangements of the labeled macromolecule affecting its correlation time (At~a"a). I. 3. Determination of the distance between parama#netic centres localised on the protein molecule There are techniques permitting the distance between the nitroxyl radical and the paramagnetic metal ion (Taylor et al., 1969; K~iiv~ir~iinen et al., 1972; Lichenstein, 1968) or two nitroxyl radicals (Kulikov et al., 1972; Kokorin et al., 1972) to be evaluated. These techniques are based either on the dependence of line splitting or additional widening upon the distance between paramagnetic centres due to the interaction of magnetic dipoles (Falle et al., 1966; Abragam, 1961) or on the dependence of the resulting ESR spectrum upon the magnitude of high frequency signals detector current (saturation curve) (Kotel'nikova et al., 1973; Kulikov, 1976). (a)

II. A P P L I C A T I O N O F S P I N - L A B E L M E T H O D FOR THE STUDY OF ANTIBODY C O M B I N I N G SITES

II. 1. Study of rigidity and depth of combining sites The first to use spin-labeled hapten to study antibody combining sites were Stryer and Griffith (1965). The hapten consisted of 2,4-dinitrophenol (DNP) conjugated with piperidine iminoxyl derivative (Fig. 3). The formation of a specific complex with anti-DNP results in a strong immobilisation of spin-labeled hapten. The restricted rotational freedom of hapten in the cavity of combining site may be caused by steric hindrance as well as by hydrophobic and van der Waals interactions, thus pointing to considerable structural rigidity of combining sites. Using mono- and bivalent spin-labeled DNP haptens Hsia and Piette (1969) studied the depth and heterogeneity of combining sites of rabbit anti-DNP antibodies. The authors investigated the dependence of correlation time of spin-labeled monovalent D N P hapten bound to antibody combining site upon H + d, with H being the dimension of the hapten as such and d being the distance between hapten and the label that can be changed by lengthening the hydrocarbon chain between them. Changing H + d from 10 to 12 ~ was found to lead to a strong decrease in the label correlation time that remained further almost without alteration. Such a decrease is accounted for by the fact that over the above range H + d is exceeding the depth of the combining site, and the label escapes from it, thus acquiring freedom of rotation. It follows therefore that the depth of the combining site is equal to 1~12 A.. Similar data were obtained for experiments with bivalent haptens. Addition of bivalent hapten to antibodies revealed a strongly immobilized ESR spectrum due to the formation of antibody dimers or trimers. The minimum length of bivalent hapten still retaining the property to bind 2 antibody molecules was 21 A. It is thus seen that the minimum depth of the combining site obtained by this approach is 10.5 A. The dimensions of combining sites appear to be very little dependent on the species specificity. Thus, (c)

(b)

I t I~

B Ama ~

-',"t

i I

I

Fig. 2. ESR spectra of spin-labels: (a) free rotation in solution (correlation time ~ t 0 -11 sec); (b) immobilized label (correlation time ~ 10-7 see); (c) ESR spectra of spin-labels, existing in two different mieroenvironment. Areax is a function of correlation time and N-O group environment polarity.

Spin-label Studies of Immunoglobulin Structure

CH 3 CH 3

NO z

Fig. 3. Spin-labeled hapten (DNP nitroxid) (Stryer & Griffith, 1965).

for example, the same procedure of successive increase in the distance between DNP group and spinlabel showed the depth of combining sites of chicken anti-DNP antibodies and mouse myeloma IgA MOPC 315 with a strong affinity to DNP to be 10 4- I A (Piette et al., 1971). A similar result was reported by Dwek et al. (1975) from their structural study of the combining site of protein 315 Fv fragment by means of spin-labeled haptens. In accord with X-ray evidence on the Fab fragment of McPC 603 protein the dimensions of the combining site were as follows: depth 12 A, width--15 A, length-- 20A (Segal et al., 1974). II. 2. Di~'rence in combining site properties after primary and secondary immune response The spin-label method allowed also differences in properties of rabbit antibodies to 2,4,6-trinitrophenyl (TNP) after primary and secondary immunisation to be elucidated (Hsia & Little, 1971). These antibodies were studied by means of spin-labeled TNP hapten with a different spatial structure both by ESR and quenching of antibody fluorescence. Antibodies formed during primary and secondary immune response were found to have a different structure of combining sites. Complexes of primary antibodies with haptens were less rigid and more sensitive to organic solvents and steric ligand changes. II. 3. Study of combining sites in antibodies to charged haptens Piette et al. (1972) described the behaviour of complexes of negatively charged spin-labeled benzoate and positively charged spin-labeled trimethylphenylammonium with corresponding rabbit antibodies. According to ESR data antibody complexes with negatively charged spin-labeled haptens were more rigid than those with positively charged spin-labeled haptens. It was also shown that the extent of immobilisation of the N - O group of spin-labeled benzoate in antibody combining sites was different depending on rabbits under study. 1I. 4. Study of the cross reactivity of antibodies by spinlabeled haptens Hsia and Piette (1969) reported on the cross reactivity and structural heterogeneity of rabbit anti-DNP. The authors compared the ESR spectra of complexes of spin-labeled DNP (DNP-SL) with antibodies isolated by means of homologous (AB--D) and cross reactive (AB-T) haptens (Fig. 4). Am,. for the complex (AB-D + DNP-SL) was thereby found to be 59G and that for (AB T + DNP-SL) 62G. AreaXis the function of the correlation time and polarity of the environment of the N - O group (Hamilton & McConnel, 1968). Therefore the difference in these ESR spectra

1003

may be due either to stronger immobilization of the group in AB-T + DNP-SL or its more hydrophobic environment. In either case, these differences disclose structural differences in combining sites of AB-D and AB-T antibodies. Another experimental series involved AB-D complexes with homologous DNP-SL and cross reactive spin-labeled haptens: ONP-SL and PNP-SL (Fig. 4). Am,x of ESR spectra for cross reactive spin-labeled haptens, ONP-SL and PNP-SL, located in combining sites were also found to exceed those for homologous DNP-SL. The authors explained this result as due to the latter hapten being located in the combining site so that the label N - O group did not directly interact with its amino acid residues. On the other hand, the cross reactive hapten formed a less rigid complex whose label had a greater freedom of rotation, thus enabling it to interact with hydrophobic regions of combining sites. ESR spectra of spin-labeled cross reactive haptens differed also from those of homologous DNP-SL by having components corresponding to faster rotation. This could result either from the equilibrium existing between 2 orientations pf spin-labeled haptens in combining sites or from different affinity in various antibody populations, specific to the respective hapten. Hsia & Little (1973) investigated the interaction of two spin-labeled haptens: DNP-hydrazone derivative of 1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidone (I) and N(l-oxyl-2,2,5,5-tetramethyl-3-methyl aminopyrrolidinyl)2,4-dinitrobenzene (II) with two myeloma Ig (MOPC 315 and 460) by means of ESR and quenching of protein fluorescence. They found that N - O groups of I and II bound to protein 315 are in a strongly immobilized state but ESR spectrum of II reveals two types of immobilized components as distinct from that of ESR of I. On protein 460 the rotational freedom of the N - O group of 1 is also rather limited whereas that of the group of II remains practically unaffected. Yet the affinity of I and II to 315 and 460 proteins is very close as judged by rather similar quenching of protein fluorescence on complexation with spin-labeled haptens and by thermodynamic evidence on this process. It follows therefore that N6a

O~N--~NH -

~

CI)

I. 0

I° 0 Fig. 4. Hqmologous (I) and cross-reactwe (I1 and lid spinlabeled haptens (Hsia & Piette, 1969). (I) DNP-SL; (II) ONP-SL; (III) PNP-SL.

1004

A.I.K.~IV.~,R.~INEN and R. S. NEZLIN

the immobilization extent of the N - O group of spinlabeled hapten is not always characteristic of the stability of the complex. The authors suggest the difference in the extent of immobilization of I and II as due to two causes. First, ligands are bonded in different subsites of a conformationally rigid combining site. Secondly, the conformation of the combining site may be more or less stabilised depending on the differences in the ligand structure. Another work 0Nong et al., 1974) deals with the study the causes leading to the appearance of two types of immobilized components a and b in ESR of complex II with protein 315 (Fig. 5). It is shown that these 2 components are due to 2 enantiomeric forms of spin-labeled hapten II. The N - O group of one ligand form is thereby in a less hydrophobic, that of another ligand form in a more hydrophobic microenvironment, thus ~ausing a different position of immobilized components on the tension axis of the magnetic field. The binding of separated enantiomeric spin-labeled amines + DNP with protein 315 give rise to components of only one type (Figs. 5B and C). II. 5 Practical application of spin-labeled haptens Substitution of spin-labeled haptens in antibody combining sites by homologous haptens was made use of by Leute et al. (1972a,b) to estimate morphine concentration in the solution under study. This method used the effect of a strong increase in the amplitude of ESR spectrum bands of spin-labeled morphine on its transition from immobilized to free

a.b

I

i

I

/

I I I I

I I I i

b a

I I

I I I I

I

I

I

I

I

/]

s

'i

'i

^ I

/I

I

I v

, I

~

v

I

It

lOG

Fig. 5. ESR spectra of complexes of IgA myeloma protein 315 with spin-labeled hapten (A) and its separated enantiomeric forms (B and C) (Wong et al., 1974).

state owing to its displacement from the combining site. In the last case the amplitude of narrow bands accounted for morphine concentration in the solution in question with the lower limit of sensitivity of the method being 10-7M morphine (0.03g/ml). Hsia et al. (1973} used a complex of [N-(l-oxyl-2,2,5,5-tetramet hyl- 3- met hylaminopyrrolidinyl)-2,4-d i n it r obenzene] with myeloma lgA MOPC 315 as a reference system to study the cross reactivity of this protein in respect to DNP and its analogs. The amount of spin-labeled hapten displaced from combining sites by non-labeled hapten was determined by the intensity of the high-field ( - 1) component in ESR spectrum. Quantitatively, the method gave results in good accord with those obtained by equilibrium dialysis. The method is thus considered as very promising for immunochemical studies.

111. USE OF SPIN-LABELS FOR T H E STUDY OF T H E FLEXIBILITY OF I M M U N O G L O B U L I N MOLECULES

III. 1. Determination of correlation time by means of spin-labeled haptens Valentine and Green found by electron microscopy (1967) that the angle between Fab fragments of IgGantibodies can vary over a large range. It was subsequently shown by physicochemical methods that flexibility was common in a varying degree to all Ig molecules (Nezlin et al., 1973: Zagyansky et al.. 1969; Yguaribide et al.. 1970; K~iivar~iinen et al., 1973, 1974; Nezlin et al., 1970). The main approach used thereby involved the comparison of experimental correlation time of Ig molecule with the calculated one in terms of the assumption on the rigidity of the molecule. Thus, if IgG molecule is considered as a rigid rotation ellipsoid with 1:2 axis ratio and mol. wt of 150,000, its correlation time should be 73 nsec, whereas the experimental value was, as a rule, considerably lower: 20--40 nsec. This shows the fragments capable of independent relative motion and points to the flexibility of parts of peptide chains connected them. In order to determine the correlation time of spinlabeled hapten complexed with antibody Stryer and Griffith (1965) studied the behaviour of substances containing both the fluorochrome group and the spin label. Changing the viscosity of its solution by adding glycerine they made ESR spectrum similar to that of spin-labeled hapten localized in the combining site. Then the correlation time of this substance was evaluated by fluorescence polarisation. The value obtained by this approach for the spin-labeled hapten complexed with antibody was found to be 12nsec. The authors were correct in suggesting that correlation time of Fab may be bigger since the possible rotation of nitroxyl ring relative to DNP could not be excluded. Such a drawback is general for all attempts to evaluate the correlation time of Fab fragment of IgG by means of spin-labeled hapten. To determine the correlation time of spin-labeled hapten localised in the combining site, Hsia and Piette (1969) plotted the calibration dependence of ESR spectra on the viscosity of spin-label solution. Correlation time (z) corresponding to a particular spectrum was determined by Stokes-Einstein law equation (1), taking the effective Stokes radius of the

Spin-label Studies of Immunoglobulin Structure label as 5 A. Correlation time of the shortest spinlabeled hapten, most rigidly reacted with the combining site, was thereby calculated to be 39 nsec. Dwek et al. (1975) using the method of Shimshick & McConnel (1972) calculated the correlation time of the spin-labeled DNP localized in the combining sites of IgA (MOPC 315) and its Fab and Fv fragments and found them to be 44.4, 23 and 6.5 nsec, respectively. III. 2. Determination of correlation time by means of spin-labels localised in antibody combining sites In contrast to the above studies we have obtained rabbit antibodies directly to the spin-label 2,2,6,6tetramethyl- peperidine-4-amino- (N - dichlorotriazine) used as a hapten during immunization (Kiiiv~iriiinen et al., 1973; Kiiiv~iriiinen et al., 1974). The formation of specific complexes of antibodies, their pepsin and papain fragments with the spin-label resulted in its strong immobilization (Fig. 6). Mixing corresponding quantities of solutions of non-specific rabbit IgG and label under similar conditions led only to the appearance of ESR spectrum for the free label. The correlation time of labels was estimated according to McCalley et al. (1972). For spin-label complexed with antibodies, F(ab')2, and Fab (at pH 6.3) it proved to be 32. 30 and 18 nsec, respectively. Our correlation time data for IgG and Fab are in good agreement with the data of Dwek et al., 1975 (see Section III. 1.). The difference in correlation time estimated for the Fab either in a free state or in F(ab')2 and intact antibody pointed to the restricted rotational freedom of Fab in last cases. Higher temperatures gave rise to decreased correlation time as reduced to standard conditions (rl/T = 3.105P/deg) of label complexes with F(ab')2 and IgG. It could thus be suggested that heating led to greater freedom in relative motion of their subunits. IlL 3. Comparative study of IgG and IgE flexibility by means of labels non-riglidly bonded outside combining site We evaluated the proper correlation time of spin labels covalently bounded to IgG and myeloma human IgE (Yu) and the correlation time of fragments

1005

as such in intact molecules (zM) by the method described in section I. 2 (Nezlin et al., 1973; K~iiviiriiinen, 1975). zR for IgG and IgE was thereby found to be 9 and 8.5 nsec, and zu estimated 35 and 60 nsec, respectively, r M obtained for IgG (35 nsec) was in good accord with zM for rabbit IgG (32 nsec) as determined by the method of rigidly bound label (see section III. 2). At the same time rg for these proteins was evaluated by fluorescence polarization (Nezlin et al., 1973). The correlation time obtained by the two methods proved to be rather similar. It followed therefore that the relative motion of IgE subunits was less free than that of Ig subunits owing either to additional interaction between IgE subunits as compared to IgG or to the special nature of S-S bridges of E-chains (Bennich, 1974). IV. MOBILITY O F D O M A I N S AND C O N F O R M A T I O N A L C H A N G E S IN Fab F R A G M E N T S O F ANTIBODIES O N INTERACTION WITH ANTIGENS

IV. 1. Analysis of ESR spectra for spin-labeled loG, F(ab')2, Fab and dimers of L chains The information obtainable from the ESR spectrum of the spin-label was dependent on site of its bonding. The particular amino acid residue (or residues) to which the label was being bonded was determined by its chemical structure and labeling conditions (e.g. pH). In our work (Kiiiviiriiinen & Nezlin, 1976) proteins were labeled with two radicals: 2,2,6,6-tetramethylpiperidine-4-amino-(N-dichloro triazine)--R 1, and N-(2,2,5,5-tetramethyl-2,5-dihydropyrrolyl-carbonyl)imidazol--R2. Labeling was effected by 3 different procedures. First, proteins were labeled with R1 at pH 9 after preliminary treatment with trichloro-Striazine at pH 7.5. It was likely that under these conditions R~ reacts with lysine residues. Secondly, proteins were labeled with Ra at pH 7.5 (0.01M Tris-HC1). In this case R1 should most likely react with histidine residues. Thirdly, proteins were labeled with R 2 at pH 7.5. In that case the most likely R2 reacts with lysine residues similarly to the first case. This ~tudy showed that ESR spectra were strongly affected by the site of label bonding (K~iiviir~iinen & Nezlin, 1976). The ESR spectrum of R t. and R 2 labels

(a)

(c)

(b)

o

+1

66.3G

ILl

I=

64..2G

---I

Fig. 6. ESR spectra of spin-label complexed with (a) intact rabbit antibodies, (b) their F(ab')2 and (c) Fab fragments (K~iviirViinen et al., 1973).

1006

A.I. KAIV)i,R,~INEN and R. S. NEZLIN

apparently conjugated with lysine residues of IgG revealed 3 narrow bands characteristic of weakly immobilized label not affected by interaction with the macromolecule. By contrast the spectra of IgG, Fab and L chains labeled by R 1 at pH 7.5 (second procedure) revealed 5 components (Fig. 7). These spectra pointed to the possibility for the label to be in 2 different microenvironments, A and B. The similarity in the shape of ESR spectra for different IgG and their subunits seemed to be due to their common structural elements, that is domains, and their interaction (K~iiviiriiinen & Nez!in, 1976; Kiiiviir~iinen et al., 1973). This suggestion was substantiated by the fact that after halving of spin-labeled L chains (K~iiv~iriiinen & Nezlin, 1976), A-components corresponding to more immobilized label state disappeared completely (Fig. 7). It was therefore of importance that according to physico-chemical and immunochemical evidence (BjiSrk et al., 1971; Vengerova et al., 1972) halves of L chains retained their main general structure they had in intact L chains. IV. 2. Interpretation of forms and changes in ESR spectra of spin-labeled immunoglobulins and their subunits The form of ESR spectra of IgG and their subunits (Fig. 7a) may be due to the following two causes and their combinations (K~iiv~iriiinen & Nezlin, 1976; K~iiv~ir~iinen et al., 1973). (1) The spectrum results from the overlapping of 2 spectra corresponding to 2 different sites of label bonding with different microenvironments. (2) ESR spectrum is determined by the capacity of each label to fluctuate between 2 different microenvironments. Such an explanation presupposes the capacity of Fab fragments and dimers of L chains to change their quarternary structure in the process of domain Brownian rotation. In that case A-components of ESR spectra correspond to a more compact A-Conformer (the label is immobilized) and B-components account for a less compact conformer (the label rotates more freely). The first explanation is not satisfactory because the interaction of spin-labeled antibody with antigen results not only in increased immobilization of label in A-state but' also in increased amount of the label

in this state (see section IV. 3). These experimental facts can be interpreted by conformer model. It is thereby enough to assume that the interaction of the combining site with the antigen determinant owing to the reorientation and stabilization of V domains leads to an equilibrium shift between A- and B-conformers towards the A-conformer. The correctness of the conformer model is substantiated by the disappearance of A-components in ESR spectrum for spin-labeled L chains on their splitting to halves, that is when they lost the ability to be present as A-conformer (see section IV.l). We have previously suggested that stabilization and reorientation of V domains under the action of the antigen determinant induce a change in the relative motion of all other domains involved in the Ig molecule (K~iiv~ir~iinen et al., 1973). This may lead to a change not only in the conformation of Fab and Fc subunits but in their interaction as well. This suggestion is in accordance with the results of studies using such methods as small angle X-ray scattering (Pilz et al., 1973) and circular polarization of fluorescence (Shlessinger et al., 1975). IV. 3. Changes in ESR spectra of spin-labeled antibodies on interaction with antigens It was elucidated how the specific interaction of antigens with antibodies could influence on the equilibrium between more and less compact forms of Ig subunits. In these experiments several preparations of pure antibodies labeled with R1 at pH 7.5 were used: rabbit anti-human hemoglobin and anti-bovine IgG, as well as donkey anti-human IgG (Kiiiviiriiinen & Nezlin, 1976; Kiiiviiriiinen et al., 1973). Under the conditions of our experiment spin-labels were bonded essentially with Fab fragments. The nature of changes in ESR spectra for spin-labeled antibodies on their interaction with antigens was not affected by the formation of the precipitate. In all cases under study the complex formation of spinlabeled antibodies with antigens led to increased area of A-component as compared with the reference ESR spectrum. In addition, there took place a shift of A

,oG ~

B

~(b) , {lOG c)

(a)

A

Fig. 7. ESR spectrum of spin-labeled dimers of rat L chains (a); left part of the same spectrum (b) and left part of ESR spectrum of halves of spin-labeled rat L chains (c). Chains were labeled by R1 at pH 7.5 (K~iiviiriiinen & Nezlin, 1976).

Spin-label Studies of Immunoglobulin Structure components corresponding to increased immobilization of the label in A-state. As the area of A-components was determined by the amount of label in this state, its increase means increased amount of label in A-state. CONCLUSIONS The spin-label method has already provided considerable information on the structure of immunoglobulins, which is in good agreement with X-ray crystallographic "evidence (Davies et al., 1975; Poljak, 1975). In particular, a good correlation between the data on the depth of antibody combining sites is provided by means of spin-labeled haptens and X-ray structural analysis. It is, however, evident that the spin-label approach is most useful for the study of dynamic properties of protein structure owing to the limitations of X-ray crystallography. The analysis of spectra of spin-labeled Ig and their subunits suggests that globulae-domains of peptide chains are capable of relative motion. The reported data obtained by means of spin-labels indicate that the formation of antibody complexes with antigens makes Fab fragments of antibodies more compact at the expense of the change in the nature of the relative motion of domains. It can be thus concluded that the spin-label approach can provide valuable information for the elucidation of the controversial problem of conformational changes in antibodies as a result of interaction with antigens. Acknowledgements--The research in the authors' laboratory is supported by WHO. We would like to express our thanks to Prof. A. L. Pumpiansky for the translation of the article.

REFERENCES

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