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Studies on waldenström macroglobulins III. The conformation of immunoglobulin M investigated by infrared spectroscopy
BIOCHIMICA ET BIOPHYSICA ACTA
655
BBA 35854 S T U D I E S ON WALDENSTROM MACROGLOBULINS I I I . T H E CONFORMATION OF IMMUNOGLOBULIN M I N V E S T I G A T E D BY I N F R A R E D SPECTROSCOPY
J. N. M I L L E R
Department of Chemistry, Loughborough University of Technology, Leicestershire, LEII 3TU, (United Kingdom) (Received J a n u a r y I2th, 1971)
SUMMARY
The conformation of immunoglobulin M in various solvents has been investigated by studying its infra-red spectrum in the amide I' region. The results suggest that the molecule is non-helical in its native state and m a y contain the anti-parallel /?-structure. Small helical regions may occur, however, in strongly alkaline solutions or in the presence of sodium dodecyl sulphate. The conformation of the subunit of immunoglobulin M is indistinguishable from that of the whole molecule.
Recent studies of the conformation of immunoglobulin M (IgM) and other immunoglobulins by the methods of optical rotatory dispersion (ORD) 1-a and circular dichroism (CD)4,5 have shown that these molecules are largely devoid of a-helices but may contain appreciable amounts of another ordered structure. Further information on the nature of this structure might be obtained by infra-red spectroscopic measurements. By studying antigen-precipitated fibrils of human ?J-globulin in polarised light, IMAI-IORIs was able to show that their anaide I absorption maximum was at a similar frequency (I635 cm -1) to that of the anti-parallel cross-/? structure of poly-O-acetyl-L-serine: the conformation of 7-globulin in solution m a y not be the same, however. It has since been shown that studies of amide I' frequencies in 2H20 solution may give information on protein conformations v. Rabbit immunoglobulin G (IgG) and its lrat~ and Fe fragments 8, and other proteins 9-n, have been studied in this way. The present communication describes the infrared spectrum of immunoglobulin M (IgM) in the amide I' region in a variety of conditions, and also the spectrum of the subunit of IgM in the same region. IgM samples were isolated from the sera of two Waldenstr6m's maeroglobulinaemia patients as previously described 12. The subunit of IgM with its interehain Abbreviations: IgM, immunoglobulin M; lgMs, subunit of IgM; Ig(;, inlnlunoglobulin G; ORD, optical rotatory dispersion; CD, circular dichroism.
Biochim. Biophys. Acta, 236 (I97I) 655-658
656
j.N. MILLER
disulphide bonds intact, lgMs, was prepared by reducing IgM at pH 7.8 with 20 mM cysteine and alkylating with 0.05 M iodoacetamide. Unreduced IgM was removed by gel filtration on Sephadex (;-20o in phosphate-NaC1 buffer, pH 7.8, I o.2. The isolated IgMs gave a single band on cellulose acetate electrophoresis in a buffer containing 6 M urea at pH 8.6 and was not separated into its constituent polypeptide chains by gel filtration on Sephadex G-Ioo using I M propionic acid as solvent. Bovine y-globulin (Cohn fraction II of bovine plasma) was obtained from Koch-Light Ltd. Protein samples were equilibrated against the appropriate solvent by repeated dialysis over a period of several days. 2H20 of isotopic purity 99.7°//o (Prochem Ltd., Croydon, and Ryvan Chemical Co. Ltd., Botley) was used throughout, and tile solvent p2H was adjusted when necessary by the addition of 2HC1 or NaO2H. After dialysis the solvent contained sufficient HO~H to render observations in the amide I I ' region (circa I55O cm-1) impracticable, p2H values were obtained by adding 0. 4 to nominal pH-meter readings 14. Where necessary, protein solutions were concentrated to the working concentration of approx. 40 mg/ml by absorption of solvent and low molecular weight solutes with polyacrylamide granules ("Lyphogel": GehnanHawksley Ltd., Lancing). Separate experiments showed that this procedure had no effect on the observed infrared spectra in the frequency range I6OO-I7OO cm -1. Spectra were recorded on a Unicam SP2ooG grating spectrometer, operated at a scan speed of approx, o.75 cm t per sec. The wavenumber scale was calibrated in each experiment using a polystyrene film. The resolution of the spectrometer in the amide I' region was approx. 2 cm -~ and the wavenumber reproducibility approx. 7~ I cm -1. Demountable sample cells with CaF 2 windows and teflon spacers giving path lengths of o.o5 and o.Io mm (Ross Scientific Co. Ltd.) were used. The amide I' spectra of IgM samples "Man" and "Glo" and of bovine y-globulin, all dissolved in phosphate-NaC1 buffers at p2H 8.o, are shown in Fig. I (a-c). The
mm
i
b
Transmittance
I
1700
I
I
1600
cm-I
Fig. 1. Amide I' spectra in phosphate-NaCl buffers at p2H 8.0 (a) IgM "Man", (b) IgM "Glo", (c) bovine y-globulin. The absorption at 155o-16oocm 1 is due to side-chain groups7.
Biochim. Biophys. Acta, 236 (1971) 655.-658
657
WALDENSTROM MACROGLOBULIN
close similarity between the spectra may be compared with ttle similar ORD properties of these proteins a. The principle features of each infrared spectrum are the absorption maximum at 1644 ~: I cm -x and a second less intense absorption maximum at 1635 :E I cm -1. There are also shoulders at approx. 1616, 1629 and 1685 cm 1. The I644-cm -1 absorption band is characteristic of the spectra of disordered proteins and polypeptides in 2H20 solution. The absorption at 1635 cm -1 is close to the absorption frequency of deuterated polypeptides having the fl-conformation 1~ and has also been observed in proteins which contain some /5-structureL There is no absorption maximum or shoulder apparent at 165o cm -1, the amide I' frequency characteristic of the a-helix. Therefore, in agreement with the interpretation of ORD data, the infrared spectra suggest that the secondary structure of IgM is effectively non-helical, and contains a mixture of disordered and fi-struetures. The presence of the 1685 cm -1 shoulder suggests that it is the anti-parallel/5-structure which is present as theoretical studies indicate that the parallel /5-structure should not show this feature ~. Some features of the spectra, however, indicate that this interpretation must be treated with caution. The shoulder at 1685 cm 1 is difficult to detect: it was apparently not observed by ABATUROVet al. in IgG. On the other hand, the shoulder at 1668 cm I observed by those workers in the spectrum of IgG could not be detected in the IgM spectrum. The significance of the absorption at 1635 cm -~ is also uncertain, since small features at this frequency have been observed in the spectrum of soyabean trypsin inhibitor, which probably has a disordered conformation 17, and in the spectra of other proteins believed not to contain/5-structures is. Finally the shoulders at frequencies below 163o cm -~ are difficult to interpret. The/5-structures of some homopolypeptides have absorption bands between 161o and 163o c m - ' (see refs. 19 and 20) and small features have also been observed in this region of the spectra of cytochrome c21 and methanol-denatured fl-lactoglobulin 7. I
t
~m oR
Tronsrnittonc¢
i
1700
i
I 1600
cm-I
Fig. 2. Amide 1' spectra of IgM "Man" in (a) phosphate-NaC1 buffer p2H 8.0 containing 0.06 M sodium dodecyl sulphate, and (b) in phosphate-NaC1 buffer adjusted to p2H 11.8 with NaO2H.
Biochim. Biophys..qcta, 236 (I97 I) 655-658
058
J.X.
MILLER
The infrared spectrum of IgM " M a n " at p"H 4.7 was identical to the spectrum obtained at pYH 8.o ORD data also indicate that no substantial conformation change occurs over the pH range 4.5--7.8 ~2. At pYH I1.8 however, a small shoulder appears in the spectrum at 165o cm 1, suggesting that in alkaline solutions short lengths of a-helix may form in IgM (Fig, 2(a)). The Moffit-Yang b0 parameter of IgM at pH 12 also indicates the presence of small amounts of this structure 2a. The spectrum of IgM at pYH 1. 9 could not be observed because of the insolubility of the protein at this pYH value, which contrasts with its readv solubility in o.o5 M HC1 in HyO at pH 1. 4. The spectrum of IgM "Man" at pYH 8.o in a buffer containing o.o6 M sodium dodecyl sulphate is shown in Fig. 2(b). The absorption maxima previously described are still observed, but there is also a pronounced shoulder at 165o-1657 cm -1, suggesting that considerable quantities of a-helix may have been formed. The effect of detergents on protein conformations is rather variable 24, but JIROENSO~XS et al. 2~ showed that decyl sulphate promoted R-helix formation in Bence-Jones proteins, which are homogeneous immunoglobulin light chains. The spectrum of IgMs "Man" was found to be identical to that of the whole molecule (Fig. z(a)), lending further support to the view that reduction of the intersubunit disulphide bands of IgM does not alter the conformation of the polypeptide chains 3. Infrared spectroscopy in the amide I' region therefore suggests that the native conformation of IgM is non-helical, and that the ordered structure present may be of the antiparallel fi-type. The spectra obtained in a variety of conditions support the findings of ORD and CD experiments and confirm the value of infrared studies in the study of protein conformations. Ambiguities in the interpretation of certain minor features of the spectra might be resolved by extending observations to lower frequencies, where further conformation-dependent absorption bands occur 15. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 i8 19 20 21 22 23 24 25
JOHNSON AND J. N. MILLER, Chem. Soc. Special Publ., 23 (I968) 59. J. DORRINGTON AND C. TANFORD, J. Biol. Chem., 243 (I968) 4745. JOHNSON AND J. N. MILLER, Biochim. Biophys. Acta, 2o 7 (I97 o) 308. E. CATHOU, A. KULCZYCKI, JR AND E. I-]ABER, Biochemistry, 7 (I968) .3958. Do1 AND B. JIRGENSONS, Biochemistry, 9 (I97O) io66. IMAHORL Biopolymers, I (I963) 563. Sus1, S. N. TIMASHEFF AND L. STEVENS, J. Biol. Chem., 242 (1967) 546o, 5467 . V. ABATUROV, R. S. NEZLIN, T. I. VENGEROVA AND J. M. VARSHAVSKY, Biochim. Biophys. Acta, 194 (I969) 386. D. F. H. WALLACH, J. M. GRAHAM AND g . R. FERNBACH, Arch, Biochem. Biophys., 131 (1969) 322. t . V. ABATUROV AND J. M. VARSHAVSKY, Studia Biophysica, * 3 (1969) 47. g. V. ABATUROV, U. M. AzIzov, B. J. ROSLYAKOV ANI) [1. I. I'¢HURGIN, Biofizika, 14 (I969) 743. P. JOHNSON AND J. N. MILLER, Biochim. Biophys. Acta, 2o 7 (197o) 297. ]7). BEALE AND A. FEINSTEIN, Biochem. J., 112 (i969) 187. p. K. GLASOE AND F. A. LONG, J. Phys. Chem., 64 (I96O) 188. -~. MASUDA, K. FUKUSHIMA, T. FUJi1 AND T. MIYAZA~VA, Biopolymers, 8 (1969) 91. T. MIYAZAWA AND E. R. BLOUT, J. Chem. Phys., 83 (1961) 712. B. JIRGENSONS, M. I~A~VABATAAND S. CAPETILLO, Die Mahromol. Chem., 125 (1969) 126. J. N. MILLER, to be published. D. K. SARKAR AND P. DOTY, Proc, Nat. Acad. Sci., U.,~., ,55 (1966) 981. S. IKRDA, Biopolymers, 5 (I967) 359D. D, ULMER AND J. H. R. KAGI, Biochemistry, 7 (1968) 27I° . J. N. MILLER, P h . D . Thesis, U n i v e r s i t y of Cambridge, 1968. P. CALLAGHAN AND N. H. MARTIN, Biochim. Biophys. Acta, 79 (1964) 539B. JIRGENSONS AND S. CAPETILLO, Biochim. Biophys. Acta, 214 (197 o) i. B. JIRGENSONS, S. SAINE AND D. L. R o s s , ,[. Biol, Chem., 241 (1966) 2314. P, K. p, R. E. K. H. L.
Bioehim, Biophys. Acta, 236 (I97 I) 655 658
655
BBA 35854 S T U D I E S ON WALDENSTROM MACROGLOBULINS I I I . T H E CONFORMATION OF IMMUNOGLOBULIN M I N V E S T I G A T E D BY I N F R A R E D SPECTROSCOPY
J. N. M I L L E R
Department of Chemistry, Loughborough University of Technology, Leicestershire, LEII 3TU, (United Kingdom) (Received J a n u a r y I2th, 1971)
SUMMARY
The conformation of immunoglobulin M in various solvents has been investigated by studying its infra-red spectrum in the amide I' region. The results suggest that the molecule is non-helical in its native state and m a y contain the anti-parallel /?-structure. Small helical regions may occur, however, in strongly alkaline solutions or in the presence of sodium dodecyl sulphate. The conformation of the subunit of immunoglobulin M is indistinguishable from that of the whole molecule.
Recent studies of the conformation of immunoglobulin M (IgM) and other immunoglobulins by the methods of optical rotatory dispersion (ORD) 1-a and circular dichroism (CD)4,5 have shown that these molecules are largely devoid of a-helices but may contain appreciable amounts of another ordered structure. Further information on the nature of this structure might be obtained by infra-red spectroscopic measurements. By studying antigen-precipitated fibrils of human ?J-globulin in polarised light, IMAI-IORIs was able to show that their anaide I absorption maximum was at a similar frequency (I635 cm -1) to that of the anti-parallel cross-/? structure of poly-O-acetyl-L-serine: the conformation of 7-globulin in solution m a y not be the same, however. It has since been shown that studies of amide I' frequencies in 2H20 solution may give information on protein conformations v. Rabbit immunoglobulin G (IgG) and its lrat~ and Fe fragments 8, and other proteins 9-n, have been studied in this way. The present communication describes the infrared spectrum of immunoglobulin M (IgM) in the amide I' region in a variety of conditions, and also the spectrum of the subunit of IgM in the same region. IgM samples were isolated from the sera of two Waldenstr6m's maeroglobulinaemia patients as previously described 12. The subunit of IgM with its interehain Abbreviations: IgM, immunoglobulin M; lgMs, subunit of IgM; Ig(;, inlnlunoglobulin G; ORD, optical rotatory dispersion; CD, circular dichroism.
Biochim. Biophys. Acta, 236 (I97I) 655-658
656
j.N. MILLER
disulphide bonds intact, lgMs, was prepared by reducing IgM at pH 7.8 with 20 mM cysteine and alkylating with 0.05 M iodoacetamide. Unreduced IgM was removed by gel filtration on Sephadex (;-20o in phosphate-NaC1 buffer, pH 7.8, I o.2. The isolated IgMs gave a single band on cellulose acetate electrophoresis in a buffer containing 6 M urea at pH 8.6 and was not separated into its constituent polypeptide chains by gel filtration on Sephadex G-Ioo using I M propionic acid as solvent. Bovine y-globulin (Cohn fraction II of bovine plasma) was obtained from Koch-Light Ltd. Protein samples were equilibrated against the appropriate solvent by repeated dialysis over a period of several days. 2H20 of isotopic purity 99.7°//o (Prochem Ltd., Croydon, and Ryvan Chemical Co. Ltd., Botley) was used throughout, and tile solvent p2H was adjusted when necessary by the addition of 2HC1 or NaO2H. After dialysis the solvent contained sufficient HO~H to render observations in the amide I I ' region (circa I55O cm-1) impracticable, p2H values were obtained by adding 0. 4 to nominal pH-meter readings 14. Where necessary, protein solutions were concentrated to the working concentration of approx. 40 mg/ml by absorption of solvent and low molecular weight solutes with polyacrylamide granules ("Lyphogel": GehnanHawksley Ltd., Lancing). Separate experiments showed that this procedure had no effect on the observed infrared spectra in the frequency range I6OO-I7OO cm -1. Spectra were recorded on a Unicam SP2ooG grating spectrometer, operated at a scan speed of approx, o.75 cm t per sec. The wavenumber scale was calibrated in each experiment using a polystyrene film. The resolution of the spectrometer in the amide I' region was approx. 2 cm -~ and the wavenumber reproducibility approx. 7~ I cm -1. Demountable sample cells with CaF 2 windows and teflon spacers giving path lengths of o.o5 and o.Io mm (Ross Scientific Co. Ltd.) were used. The amide I' spectra of IgM samples "Man" and "Glo" and of bovine y-globulin, all dissolved in phosphate-NaC1 buffers at p2H 8.o, are shown in Fig. I (a-c). The
mm
i
b
Transmittance
I
1700
I
I
1600
cm-I
Fig. 1. Amide I' spectra in phosphate-NaCl buffers at p2H 8.0 (a) IgM "Man", (b) IgM "Glo", (c) bovine y-globulin. The absorption at 155o-16oocm 1 is due to side-chain groups7.
Biochim. Biophys. Acta, 236 (1971) 655.-658
657
WALDENSTROM MACROGLOBULIN
close similarity between the spectra may be compared with ttle similar ORD properties of these proteins a. The principle features of each infrared spectrum are the absorption maximum at 1644 ~: I cm -x and a second less intense absorption maximum at 1635 :E I cm -1. There are also shoulders at approx. 1616, 1629 and 1685 cm 1. The I644-cm -1 absorption band is characteristic of the spectra of disordered proteins and polypeptides in 2H20 solution. The absorption at 1635 cm -1 is close to the absorption frequency of deuterated polypeptides having the fl-conformation 1~ and has also been observed in proteins which contain some /5-structureL There is no absorption maximum or shoulder apparent at 165o cm -1, the amide I' frequency characteristic of the a-helix. Therefore, in agreement with the interpretation of ORD data, the infrared spectra suggest that the secondary structure of IgM is effectively non-helical, and contains a mixture of disordered and fi-struetures. The presence of the 1685 cm -1 shoulder suggests that it is the anti-parallel/5-structure which is present as theoretical studies indicate that the parallel /5-structure should not show this feature ~. Some features of the spectra, however, indicate that this interpretation must be treated with caution. The shoulder at 1685 cm 1 is difficult to detect: it was apparently not observed by ABATUROVet al. in IgG. On the other hand, the shoulder at 1668 cm I observed by those workers in the spectrum of IgG could not be detected in the IgM spectrum. The significance of the absorption at 1635 cm -~ is also uncertain, since small features at this frequency have been observed in the spectrum of soyabean trypsin inhibitor, which probably has a disordered conformation 17, and in the spectra of other proteins believed not to contain/5-structures is. Finally the shoulders at frequencies below 163o cm -~ are difficult to interpret. The/5-structures of some homopolypeptides have absorption bands between 161o and 163o c m - ' (see refs. 19 and 20) and small features have also been observed in this region of the spectra of cytochrome c21 and methanol-denatured fl-lactoglobulin 7. I
t
~m oR
Tronsrnittonc¢
i
1700
i
I 1600
cm-I
Fig. 2. Amide 1' spectra of IgM "Man" in (a) phosphate-NaC1 buffer p2H 8.0 containing 0.06 M sodium dodecyl sulphate, and (b) in phosphate-NaC1 buffer adjusted to p2H 11.8 with NaO2H.
Biochim. Biophys..qcta, 236 (I97 I) 655-658
058
J.X.
MILLER
The infrared spectrum of IgM " M a n " at p"H 4.7 was identical to the spectrum obtained at pYH 8.o ORD data also indicate that no substantial conformation change occurs over the pH range 4.5--7.8 ~2. At pYH I1.8 however, a small shoulder appears in the spectrum at 165o cm 1, suggesting that in alkaline solutions short lengths of a-helix may form in IgM (Fig, 2(a)). The Moffit-Yang b0 parameter of IgM at pH 12 also indicates the presence of small amounts of this structure 2a. The spectrum of IgM at pYH 1. 9 could not be observed because of the insolubility of the protein at this pYH value, which contrasts with its readv solubility in o.o5 M HC1 in HyO at pH 1. 4. The spectrum of IgM "Man" at pYH 8.o in a buffer containing o.o6 M sodium dodecyl sulphate is shown in Fig. 2(b). The absorption maxima previously described are still observed, but there is also a pronounced shoulder at 165o-1657 cm -1, suggesting that considerable quantities of a-helix may have been formed. The effect of detergents on protein conformations is rather variable 24, but JIROENSO~XS et al. 2~ showed that decyl sulphate promoted R-helix formation in Bence-Jones proteins, which are homogeneous immunoglobulin light chains. The spectrum of IgMs "Man" was found to be identical to that of the whole molecule (Fig. z(a)), lending further support to the view that reduction of the intersubunit disulphide bands of IgM does not alter the conformation of the polypeptide chains 3. Infrared spectroscopy in the amide I' region therefore suggests that the native conformation of IgM is non-helical, and that the ordered structure present may be of the antiparallel fi-type. The spectra obtained in a variety of conditions support the findings of ORD and CD experiments and confirm the value of infrared studies in the study of protein conformations. Ambiguities in the interpretation of certain minor features of the spectra might be resolved by extending observations to lower frequencies, where further conformation-dependent absorption bands occur 15. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 i8 19 20 21 22 23 24 25
JOHNSON AND J. N. MILLER, Chem. Soc. Special Publ., 23 (I968) 59. J. DORRINGTON AND C. TANFORD, J. Biol. Chem., 243 (I968) 4745. JOHNSON AND J. N. MILLER, Biochim. Biophys. Acta, 2o 7 (I97 o) 308. E. CATHOU, A. KULCZYCKI, JR AND E. I-]ABER, Biochemistry, 7 (I968) .3958. Do1 AND B. JIRGENSONS, Biochemistry, 9 (I97O) io66. IMAHORL Biopolymers, I (I963) 563. Sus1, S. N. TIMASHEFF AND L. STEVENS, J. Biol. Chem., 242 (1967) 546o, 5467 . V. ABATUROV, R. S. NEZLIN, T. I. VENGEROVA AND J. M. VARSHAVSKY, Biochim. Biophys. Acta, 194 (I969) 386. D. F. H. WALLACH, J. M. GRAHAM AND g . R. FERNBACH, Arch, Biochem. Biophys., 131 (1969) 322. t . V. ABATUROV AND J. M. VARSHAVSKY, Studia Biophysica, * 3 (1969) 47. g. V. ABATUROV, U. M. AzIzov, B. J. ROSLYAKOV ANI) [1. I. I'¢HURGIN, Biofizika, 14 (I969) 743. P. JOHNSON AND J. N. MILLER, Biochim. Biophys. Acta, 2o 7 (197o) 297. ]7). BEALE AND A. FEINSTEIN, Biochem. J., 112 (i969) 187. p. K. GLASOE AND F. A. LONG, J. Phys. Chem., 64 (I96O) 188. -~. MASUDA, K. FUKUSHIMA, T. FUJi1 AND T. MIYAZA~VA, Biopolymers, 8 (1969) 91. T. MIYAZAWA AND E. R. BLOUT, J. Chem. Phys., 83 (1961) 712. B. JIRGENSONS, M. I~A~VABATAAND S. CAPETILLO, Die Mahromol. Chem., 125 (1969) 126. J. N. MILLER, to be published. D. K. SARKAR AND P. DOTY, Proc, Nat. Acad. Sci., U.,~., ,55 (1966) 981. S. IKRDA, Biopolymers, 5 (I967) 359D. D, ULMER AND J. H. R. KAGI, Biochemistry, 7 (1968) 27I° . J. N. MILLER, P h . D . Thesis, U n i v e r s i t y of Cambridge, 1968. P. CALLAGHAN AND N. H. MARTIN, Biochim. Biophys. Acta, 79 (1964) 539B. JIRGENSONS AND S. CAPETILLO, Biochim. Biophys. Acta, 214 (197 o) i. B. JIRGENSONS, S. SAINE AND D. L. R o s s , ,[. Biol, Chem., 241 (1966) 2314. P, K. p, R. E. K. H. L.
Bioehim, Biophys. Acta, 236 (I97 I) 655 658