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Carbohydrate-protein complex-formation
89
CARBOHYDRATE-PROTEIN
COMPLEX-FORPrfATION
SOME FACTORS AFFECTING THE INTERACTION OF D-GLUCOSE ANB mY&wCwm
%?mx
R. J. DOYLE, E. P. Pmz, Biophysics Department,
CQxCANAVALm
A
AND E. E. WOODSIDE* Roswell Park Memorial Insfirure, Buflalo, New York I4203 (U. S. A.)
(Received January 29th, 1968; in revised form, April Sth, 1968)
ABSTRACT
The effects of temperature, chelating agents, protein denaturams, and ‘alphaand beta-amylolysis on concanavalin A-polysaccharide complexes have been studied. Temperatures between 30 and 50” tend to dissociate concanavalin A-glycogen complexes, whereas temperatures above 50” denature concanavalin A solutions. The chelating agent (ethylenedinitrilo)tetraacetic acid slightly inhibits complexformation between concanavalin A and polysaccharides. Formamide, guanidine hydrochloride, 1,1,3,3-tetramethylurea, and urea competitively inhibit concanavalin A-glycogen complex-formation. Concanavalin A does not protect glycogen from glycogenolysis by alpha- or beta-amylase, although it does partially protect the ability of the alpha-amylolytic end-product to be precipitated with the protein. An ultraviolet difference-spectrum was generated by the addition of D-glucose to concanavalin A solutions, and it was characterized by inflection points at approximately 256, 264, 278, 287, and 294 nm. In contrast, the optical rotatory properties of concanavalin A solutions were not detectably altered by D-glucose. Moffitt-Yang plots for concanavalin A revealed that the helical content of concanavalin A is approximately 7.5%. Possible modes of interaction between concanavalin A and neutral sugars or polysaccharides are discussed, and it is suggested that hydrogen important role in their binding.
bonding plays an
INTRODUCTION
The reaction between concanavalin
A, a phytohemagglutinin
isolated from the
jack bean, and various polysaccharides, to form insoluble protein-polysaccharide complexes has recently received considerable attention, mainly by -Goldstein and associatesl-lO. It has been shown that concanavalin A reacts with neutral polysacchar-ides through their nonreducing chain-ends2B7-g. Several techniques. have been
employed
*Microbiology &x&x~ u. 5%.4-
for
characterizing
Depactinent,
University
concanavalin of Louisville
A-neutral
S&o01
of
D-glucan
complexes;
Medkine,
LouikviUe, Kentucky
cpibo@d.
Res., 8 (1968) 89-1~~
CARBOHYDRATE-PROTEIN
99
COhlPLExE!3
or [R’llg8, which correspond to the trough and peak mean residue rotations 1-R’1233, of the peptide bondlg. Data were obtained for [R’&33 only, as the [R’jlgs value was inaccessibIe with the instrumentation avaiIabIe. The value of [KJ,& for concanavalin A was found to be - 1830 f 10”. This value is low, and is characteristic of proteins having a low helical content (a value of - 11,m” corresponds to a completely helical poIymer)20. For the calculation of the helical content, it is necessary to determine [R&s in the completely denatured state, as well as in the native state. However, estimates of [Kj233 were inconclusive, because of the fact that concanavalin A in 8.Ohi urea exhibits a negative b, value, which indicates only a partial denaturation of concanavalin A. It is possible that the o.r.d. measurements in 8.0~ urea were biased by aggregation (cz ref. 33), although Quiocho et alp’ observed a negatiye b. value for carboxypeptidase in 8.0~ urea. Thus, it is believed that [R’J& data for concanavalin A in 8.0~ urea are unreliable. However, from the [R’& data for concanavalin A in buffer, it may be concluded that the protein has a low content of helices. Others42’43 have recently criticized the use of absolute b. values as a criterion for determining helicities, especially when low b. values are obtained. It is well known that, in some proteins, Cotton effects due to aromatic sidechains can affect the shape of the visible and near-ultraviolet 0.r.d. carves that are. generally believed to result from peptide-bond interactions&. Several investigators have studied changes in the rotatory properties of proteins in the presence of small ligands45*46. In all cases, there is little or no change in the [R’lzJ3 value when the Iigand interacts with the protein. Fasella and Hammes46 pointed out the insensitivity of the measurement in determining small conformational changes. The data reported here for concanavalin A-D-glucose support this conclusion. Atpresent, we areexamining the circular dichroism band of concanavalin A caused by L-tyrosina and L-tryptophan residues. This method should be more sensitive in detecting concanavalin A-Dglucose interactions if such interactions give rise to asymmetry around the L-tyrosine and L-tryptophan residues. The rotatory dispersion and ultraviolet difference-spectra reported here are strikingly parallel to those for biotin-avidin systems. Although b&in induces an ultraviolet difference-spectrum in avidin4’, there are no changes in its rotatory properties48. ACKNOWLEDGMENTS
The authors are indebted to Mr. F. Wissler for determining the molecular weight of concanavaIin A, and to Dr. J. BeIIo, who provided the spectropokrimeter and spectrophotometer. Financial assistance in the form of fellowships was provided by the National Institutes of Health (Grant I-Fl-GM-31, 110-01 for R. J. D., and grant GM-00718 for E. P. P-j.
1 I. J. GOLDSTEIN, C. E. HOLLERMAN, AND J. M. MERIUCK, Biochim. Biophys. Acru, 2 I. J. GOLDSTEIN, C. E. HOLLERMAN, AND E. E. SMXTH,Biocl!emistry, 4 (1965) 876.
97 (1965) 68.
Carbohyd. Res., 8 (1968) 89-100
100
R. J. DOYLE,
E. P. PlTTZ,
E. E. WOODSZDE
3 L. So AND I. J. GOLDSTEW, J. Biol. Chem., 242 (1967) 1617. I. J. GOLDSTEIN AND L. SO, Arch. Biochem. Biophys., 111 (1965) 407. B. B. L. AGRAWAL AND I. J. GOLDSIFIN, Biochim. Biophys. Acta, 133 (1967) 367. B. B. L. AGRAWAL AND I. J. GOLD.STEIN, Biochem. J., 96 (1965) 23C. I. J. GOLD-IN. R. N. IYER, E. E. SMITH, AND L. So, Biochemistry, 6 (1967) 2373. E. E. Shmm~AND I.J. GOLDSIZIN, Arch. Biochem. Biophys., 121 (1967) 88. I. J. GOLD&IN AND R. N. IYER,Biochim. Biophys. Acta, 121 (1966) 197. 10 L. So AND I. J. GOLDSITIN, .T. Immunol., 99 (1967) 158. 11 J. A. CIFONELLI AND F. SMITH, Anal. Chem., 27 (1955) 1639. 12 J. A. CIFONELLI, R. MONTGOMERY, AND F. Smm, J. Amer. Chem. Sot., 78 (1956) 2488. 4 5 6 7 8 9
13 14 15 16
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
36 37 38 39 40 41 42 43 44 45 46 47 48
D. J. MANNERS
AND
A. WRIGHT. J. Chem. Sot., (1962) 4592.
R. J. DOYLE, E. E. WOODSIDE, AND C. W. FISHEL, Biochem. J., 106 (1968) 35. M. 0. J. OLSON AND I. E. LIENER, Biochemistry, 6 (1967) 105. L. G. MOKRAXH, J. Biol. Chem., 208 (lS54) 55. T. T. HERSKOVKS AND M. LASKO~SKI, JR., J. Biol. Chem., 237 (1962) 2481. L. W. NICHOL, J. L. BETHUNE, G. KEGELES, AND E. L. HESS, in H. NEURATH (ed.), The Vol. II, Academic Press, New York, 1964, p. 305. P. UKNES AND P. Don, Aduan. Protein Chem., 19 (1961) 402. B. JIRGENSONS,Makromol. Chem., 91 (1966) 74.
Proteins,
T. C. LAURENT, Biochem. J., 89 (1963) 253.
A. LOYTER AND M. SCHRAMM, Biochim. Biophys. Acta, 65 (1962) 200. Z. &LINGER AND M. SCHRAMM, Biochem. Biophys. Res. Commun., 12 (1963) 208. R-J. DoYLE,E.E. WOODSIDEAND C. W.FISHEL, Carbohyd. Res.,5 (1967) 274. E. E. WOODSIDE, G. TROY, R. J. DOYLE, AND C. W. FISHEL, Arch. Biochem. Biophys., 117 (1966) 125. F-A. BE'ITUHEIM,T.C.LA~FZENT,AND H.PERTOFT, Carbohyd. Res.,Z (1966) 391. E. H. FISCHER AND E. A. STEIN, Enzymes, 4 (1960) 313. D. R. ROBINSON AND W. P. JENCKS, J. Amer. Chem. Sot., 87 (1965) 2462. J. BELLO, D. HAAS, AND H. R. BELLO, Biochemistry, 5 (1966) 2539. I. M. KLOTZ, Federation Proc., 24 (1965) S-24, S-33. G.G.Hm AND J. C. SWANN, Biochemistry, 6 (1967) 1591. 3. B. L. AGRAWAL AND I.J. GOLDSI-EIN, Biochim. Biophys. Acta, 147 (1967) 262. M. 0. J. OLSONAND I.E. LIENER, Biochemistry, 6 (1967) 3801. M. TORII, E. A. KABAT, AND H. J. WEIGEL, J. Immunol., 96 (1966) 797. 3. STAERK AND H. SCHLENK, Biochim. Biophys. Acta, 146 (1967) 120. A. L. GROSSBERG, G. MARKUS, AND D. PRESSMAN, Proc. Natl. Acad. Sci. US., 54 (1965) 942. G. MARKUS, Proc. Nail. Acad_ Sci. U.S., 54 (1965) 253. D. B. WETLA~FER, Adcan. Protein Chem., 17 (1962) 303. J-F. RIORDAN, M. SOKOLOVSKY, AND B. L. VALLEE, Biochemistry, 6 (1967) 358. R. D. PORETZ AND I. J. GOLDSTEIN, Abstracts, Papers, Amer. Chem. Sot. Meeting, 154 (1967) 006~. F. A. QUIOCHO, W. H. BISHOP,AND F. M. RICHARDS, Proc. NatZ. Acad. Sci. U.S., 57 (1967) 525. M. J. KRONMAN, R. BLUM, AND L. G. HOLY, Biochem. Biophys. Res. Commun., 19 (1965) 227. G. D. FAS~XAN,E. BODENHEIMER, AND C. LINDBLOW, Biochemistry, 3 (1964)1665. A. ROSENBERG, J. Biol. Chem., 241 (1966) 5119. R. E. CATHOU, G. G. HAMMES, AND P. R. SCHIMMEL, Biochemistry, 4 (1965) 2687. P. FA~ELLA AND G. G. HOLMES, Biochemistry, 4 (1965) 801. N. M. GREEN,Biochem. J., 89 (1963) 599. N. M. GREEN, Biochem. J., 89 (1963) 609.
Carbohyd. Res., 8 (1968) 89-100
CARBOHYDRATE-PROTEIN
COMPLEX-FORPrfATION
SOME FACTORS AFFECTING THE INTERACTION OF D-GLUCOSE ANB mY&wCwm
%?mx
R. J. DOYLE, E. P. Pmz, Biophysics Department,
CQxCANAVALm
A
AND E. E. WOODSIDE* Roswell Park Memorial Insfirure, Buflalo, New York I4203 (U. S. A.)
(Received January 29th, 1968; in revised form, April Sth, 1968)
ABSTRACT
The effects of temperature, chelating agents, protein denaturams, and ‘alphaand beta-amylolysis on concanavalin A-polysaccharide complexes have been studied. Temperatures between 30 and 50” tend to dissociate concanavalin A-glycogen complexes, whereas temperatures above 50” denature concanavalin A solutions. The chelating agent (ethylenedinitrilo)tetraacetic acid slightly inhibits complexformation between concanavalin A and polysaccharides. Formamide, guanidine hydrochloride, 1,1,3,3-tetramethylurea, and urea competitively inhibit concanavalin A-glycogen complex-formation. Concanavalin A does not protect glycogen from glycogenolysis by alpha- or beta-amylase, although it does partially protect the ability of the alpha-amylolytic end-product to be precipitated with the protein. An ultraviolet difference-spectrum was generated by the addition of D-glucose to concanavalin A solutions, and it was characterized by inflection points at approximately 256, 264, 278, 287, and 294 nm. In contrast, the optical rotatory properties of concanavalin A solutions were not detectably altered by D-glucose. Moffitt-Yang plots for concanavalin A revealed that the helical content of concanavalin A is approximately 7.5%. Possible modes of interaction between concanavalin A and neutral sugars or polysaccharides are discussed, and it is suggested that hydrogen important role in their binding.
bonding plays an
INTRODUCTION
The reaction between concanavalin
A, a phytohemagglutinin
isolated from the
jack bean, and various polysaccharides, to form insoluble protein-polysaccharide complexes has recently received considerable attention, mainly by -Goldstein and associatesl-lO. It has been shown that concanavalin A reacts with neutral polysacchar-ides through their nonreducing chain-ends2B7-g. Several techniques. have been
employed
*Microbiology &x&x~ u. 5%.4-
for
characterizing
Depactinent,
University
concanavalin of Louisville
A-neutral
S&o01
of
D-glucan
complexes;
Medkine,
LouikviUe, Kentucky
cpibo@d.
Res., 8 (1968) 89-1~~
CARBOHYDRATE-PROTEIN
99
COhlPLExE!3
or [R’llg8, which correspond to the trough and peak mean residue rotations 1-R’1233, of the peptide bondlg. Data were obtained for [R’&33 only, as the [R’jlgs value was inaccessibIe with the instrumentation avaiIabIe. The value of [KJ,& for concanavalin A was found to be - 1830 f 10”. This value is low, and is characteristic of proteins having a low helical content (a value of - 11,m” corresponds to a completely helical poIymer)20. For the calculation of the helical content, it is necessary to determine [R&s in the completely denatured state, as well as in the native state. However, estimates of [Kj233 were inconclusive, because of the fact that concanavalin A in 8.Ohi urea exhibits a negative b, value, which indicates only a partial denaturation of concanavalin A. It is possible that the o.r.d. measurements in 8.0~ urea were biased by aggregation (cz ref. 33), although Quiocho et alp’ observed a negatiye b. value for carboxypeptidase in 8.0~ urea. Thus, it is believed that [R’J& data for concanavalin A in 8.0~ urea are unreliable. However, from the [R’& data for concanavalin A in buffer, it may be concluded that the protein has a low content of helices. Others42’43 have recently criticized the use of absolute b. values as a criterion for determining helicities, especially when low b. values are obtained. It is well known that, in some proteins, Cotton effects due to aromatic sidechains can affect the shape of the visible and near-ultraviolet 0.r.d. carves that are. generally believed to result from peptide-bond interactions&. Several investigators have studied changes in the rotatory properties of proteins in the presence of small ligands45*46. In all cases, there is little or no change in the [R’lzJ3 value when the Iigand interacts with the protein. Fasella and Hammes46 pointed out the insensitivity of the measurement in determining small conformational changes. The data reported here for concanavalin A-D-glucose support this conclusion. Atpresent, we areexamining the circular dichroism band of concanavalin A caused by L-tyrosina and L-tryptophan residues. This method should be more sensitive in detecting concanavalin A-Dglucose interactions if such interactions give rise to asymmetry around the L-tyrosine and L-tryptophan residues. The rotatory dispersion and ultraviolet difference-spectra reported here are strikingly parallel to those for biotin-avidin systems. Although b&in induces an ultraviolet difference-spectrum in avidin4’, there are no changes in its rotatory properties48. ACKNOWLEDGMENTS
The authors are indebted to Mr. F. Wissler for determining the molecular weight of concanavaIin A, and to Dr. J. BeIIo, who provided the spectropokrimeter and spectrophotometer. Financial assistance in the form of fellowships was provided by the National Institutes of Health (Grant I-Fl-GM-31, 110-01 for R. J. D., and grant GM-00718 for E. P. P-j.
1 I. J. GOLDSTEIN, C. E. HOLLERMAN, AND J. M. MERIUCK, Biochim. Biophys. Acru, 2 I. J. GOLDSTEIN, C. E. HOLLERMAN, AND E. E. SMXTH,Biocl!emistry, 4 (1965) 876.
97 (1965) 68.
Carbohyd. Res., 8 (1968) 89-100
100
R. J. DOYLE,
E. P. PlTTZ,
E. E. WOODSZDE
3 L. So AND I. J. GOLDSTEW, J. Biol. Chem., 242 (1967) 1617. I. J. GOLDSTEIN AND L. SO, Arch. Biochem. Biophys., 111 (1965) 407. B. B. L. AGRAWAL AND I. J. GOLDSIFIN, Biochim. Biophys. Acta, 133 (1967) 367. B. B. L. AGRAWAL AND I. J. GOLD.STEIN, Biochem. J., 96 (1965) 23C. I. J. GOLD-IN. R. N. IYER, E. E. SMITH, AND L. So, Biochemistry, 6 (1967) 2373. E. E. Shmm~AND I.J. GOLDSIZIN, Arch. Biochem. Biophys., 121 (1967) 88. I. J. GOLD&IN AND R. N. IYER,Biochim. Biophys. Acta, 121 (1966) 197. 10 L. So AND I. J. GOLDSITIN, .T. Immunol., 99 (1967) 158. 11 J. A. CIFONELLI AND F. SMITH, Anal. Chem., 27 (1955) 1639. 12 J. A. CIFONELLI, R. MONTGOMERY, AND F. Smm, J. Amer. Chem. Sot., 78 (1956) 2488. 4 5 6 7 8 9
13 14 15 16
17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
36 37 38 39 40 41 42 43 44 45 46 47 48
D. J. MANNERS
AND
A. WRIGHT. J. Chem. Sot., (1962) 4592.
R. J. DOYLE, E. E. WOODSIDE, AND C. W. FISHEL, Biochem. J., 106 (1968) 35. M. 0. J. OLSON AND I. E. LIENER, Biochemistry, 6 (1967) 105. L. G. MOKRAXH, J. Biol. Chem., 208 (lS54) 55. T. T. HERSKOVKS AND M. LASKO~SKI, JR., J. Biol. Chem., 237 (1962) 2481. L. W. NICHOL, J. L. BETHUNE, G. KEGELES, AND E. L. HESS, in H. NEURATH (ed.), The Vol. II, Academic Press, New York, 1964, p. 305. P. UKNES AND P. Don, Aduan. Protein Chem., 19 (1961) 402. B. JIRGENSONS,Makromol. Chem., 91 (1966) 74.
Proteins,
T. C. LAURENT, Biochem. J., 89 (1963) 253.
A. LOYTER AND M. SCHRAMM, Biochim. Biophys. Acta, 65 (1962) 200. Z. &LINGER AND M. SCHRAMM, Biochem. Biophys. Res. Commun., 12 (1963) 208. R-J. DoYLE,E.E. WOODSIDEAND C. W.FISHEL, Carbohyd. Res.,5 (1967) 274. E. E. WOODSIDE, G. TROY, R. J. DOYLE, AND C. W. FISHEL, Arch. Biochem. Biophys., 117 (1966) 125. F-A. BE'ITUHEIM,T.C.LA~FZENT,AND H.PERTOFT, Carbohyd. Res.,Z (1966) 391. E. H. FISCHER AND E. A. STEIN, Enzymes, 4 (1960) 313. D. R. ROBINSON AND W. P. JENCKS, J. Amer. Chem. Sot., 87 (1965) 2462. J. BELLO, D. HAAS, AND H. R. BELLO, Biochemistry, 5 (1966) 2539. I. M. KLOTZ, Federation Proc., 24 (1965) S-24, S-33. G.G.Hm AND J. C. SWANN, Biochemistry, 6 (1967) 1591. 3. B. L. AGRAWAL AND I.J. GOLDSI-EIN, Biochim. Biophys. Acta, 147 (1967) 262. M. 0. J. OLSONAND I.E. LIENER, Biochemistry, 6 (1967) 3801. M. TORII, E. A. KABAT, AND H. J. WEIGEL, J. Immunol., 96 (1966) 797. 3. STAERK AND H. SCHLENK, Biochim. Biophys. Acta, 146 (1967) 120. A. L. GROSSBERG, G. MARKUS, AND D. PRESSMAN, Proc. Natl. Acad. Sci. US., 54 (1965) 942. G. MARKUS, Proc. Nail. Acad_ Sci. U.S., 54 (1965) 253. D. B. WETLA~FER, Adcan. Protein Chem., 17 (1962) 303. J-F. RIORDAN, M. SOKOLOVSKY, AND B. L. VALLEE, Biochemistry, 6 (1967) 358. R. D. PORETZ AND I. J. GOLDSTEIN, Abstracts, Papers, Amer. Chem. Sot. Meeting, 154 (1967) 006~. F. A. QUIOCHO, W. H. BISHOP,AND F. M. RICHARDS, Proc. NatZ. Acad. Sci. U.S., 57 (1967) 525. M. J. KRONMAN, R. BLUM, AND L. G. HOLY, Biochem. Biophys. Res. Commun., 19 (1965) 227. G. D. FAS~XAN,E. BODENHEIMER, AND C. LINDBLOW, Biochemistry, 3 (1964)1665. A. ROSENBERG, J. Biol. Chem., 241 (1966) 5119. R. E. CATHOU, G. G. HAMMES, AND P. R. SCHIMMEL, Biochemistry, 4 (1965) 2687. P. FA~ELLA AND G. G. HOLMES, Biochemistry, 4 (1965) 801. N. M. GREEN,Biochem. J., 89 (1963) 599. N. M. GREEN, Biochem. J., 89 (1963) 609.
Carbohyd. Res., 8 (1968) 89-100