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The conformation of amylose in neutral, aqueous salt solution
Carbohydrate
Research
349
Ekewer PubhshmmgCompany ~terdam Pnnted III Be&nun
THE CONFORMATION OF AMYLOSE IN NEUTRAL, AQUEOUS SALT SOLUTION* W
BANKS
AND
C
T
GREENWOOD
Department of Chenustry, The Umverslty of Edmbtrrgh, Edmburglr 9 (Great Brltam)
(Recewed January 2nd, 1968)
ABSTRACT
Linear amylose has been subfractlonated, and the hydrodynamxc propeties of the fractions exammed From measurements of the hmdmg-vlscoslty number, 1~1,m 0 33~ potassmm chloride, and of the weight-average molecular weight, i@,, by hght scattermg on the acetate, the relation
was found The slgmficance of the exponent m tlus relation and others m the hteratore IS dzscussed Estimations of the “stiffness” of the amylose molecule m aqueous soiutlon have been obtamed for various model systems, I e , the Kuhn statlstlcal length, the Kratky-Porod persistence length, and Nagal’s theory for a mixture of hehcal and randomly coiled segments The results of each md.vldual theory are ambiguous It 1s shown, on the basis of Nagal’s theory, that there IS defimte evidence for the absence of rlgld helical segments m amylose m aqueous solution The temperature coefficient of [?I] for amylose m aqueous solution 1s also discussed INTRODUCl-ION
The conformation of the amylose molecule m aqueous solution IS currently of much interest Although It has been suggested ’ ’ that the polysacchande undergoes a hehx-coil transformation slmllar to that of proteins, our results3 4 are not consistent with this viewpoint In tks paper, we have extended our earher hydrodynamic measurements on amylose subfractions, and have cntlcally analysed our results m terms of recent theoretical treatments which involve hehcal models EXPERIMENTAL
Linear amylose, z e , a sampIe which was completely hydrolysed mto maltose *Part 43 m the senes “Physlcochemlcal Journal, m the press
Studxs on Starches”,
for Part 42, see European Polymer
Carboh)d
Res ,
7 (1968) 349-356
AMYLOSE
355
CONFORMATION
holds down to molecular werghts of at least 1.5 x 10’ Assuming that, at thus level, R = 10 [see equation (IQ], the maximum fractron of D-glucose resrdues which can be accomodated m the hehces [calculated from equatron (II)] IS of the order of 0.4 Thus, one obtams a model whrch IS composed of essentrally equally-long segments (SO-80 monomer units) of hehx and random co11 This 1sphysrcally unreal, since there 1s no reason why the forces which stabrhzed the helix should be absent over such extended lengths In fact, the use of b, = 1.33 A, mvanably leads to a model whtch 1smconsistent with other hydrodynamic evidence We therefore suggest that, although there may be some helical character m amylose, the compact hehcal segments prevrously postulated ’ ’ do not exist, that is, the hehces are very much “looser” than has been suggested, and b, must be greater than 1 33 A The hehcal nature ofthe macromolecule in solution 1s thus not a result of mtramolecular hydrogen bonding, but merely a consequence of the a-D-(1 +4)-hnkages Indirect evidence for rigid, hehcal segments m amylose IS provided by the temperature-dependence of [q] m aqueous potassmm chloride If, as we have stressed, this 1sa &solvent for the polysacchande, [a] should be very sensmve to temperaturet’ But this IS not the case Over a range of 20-30”, there IS vutually no change m [q] Rao and Foster’ have suggested that the increase m temperature has two opposmg and equal effects the increased solvent-power of the water causes the amylose to expand, whilst the helical segments break down, making the amylose less ngrd and therefore able to take up a more compact conformatton This hypothesis implies that an increase m temperature necessarily leads to an increase m solvent-power, and that consequently solvent-solute mteractron 1s favoured at the expense of solutesolute interaction With aqueous solutrons of amylose, we suggest that the situation 1srather more complex In this case, the polymer 1sheld m solution by hydrogen bonding with the solvent, but, at the same time, polymer-polymer mteractlon 1salso dependent on hydrogen bonding As the temperature IS raised, both types of interaction will tend to become less effectrve Again, these tendencres ~111oppose each other, so that It 1s possrble for [qJ to remam unchanged over a fau-ly wide range of temperature This suggestion satlsfactorrly accounts for the solutton behavlour of amylose m aqueous potassmm chloride as r~functron of temperature zorflmtt mtroducmg a helical model We conclude that the view of amylose as a molecule composed of a number of rigid, helical segments has no experimental basis ACKNOWLEDGMENTS
We have been fortunate enough to have worked m assocratron wrth Professor Srr Edmund Hurst, C B E , F R S , for the past 16 years, and wish to express our indebtedness to him for his continued counsel and the keen interest that he has always taken in our work Thrs work was supported, m part, by a Grant from the U S Department of Agriculture, under P L 480 Carbo!zyd Res ,
7 (1968) 349-356
W. BANKS,
356
C. T
GREENWOOD
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
V S R RAO AND 3 F FOSTER, Btopofymers, 1 (1963) 527 J SZEJTLI AND S AUCXXXAT, St&he, 18 (1966) 38 W BAhKS AND C T GREENWOOD, MaXromol Chem ,67 (1963) 49 W BANKS AND C T. GREENWOOD, m Cotlfornmtron oj Btopolymers, G N RAhiAcHwmrwN (Ed ), Acadermc Press, London, 1967, p 739 W BANKS, C T GREENWOOD, AND D J HOURSTON, Trans Faraday Sor ,64 (1968) 363 R S NICGINBOTHAM AND G A MORRISON, Sh&ey Inst Mem , 22 (1948) 148 C T GREENWOOD AND J S M ROBERTSON, .I Chen? Sot , (1954) 3769 W W EVEREI-I-AND J F FOSTER, J Am Chem SOC 81 (1959) 3464 .l M G COWIE, Makromol Chem, 42 (1961) 230 W BURCHARD, Makromol Chem ,64 (1963) 110 P J FLORY, Prmczples of Polymer Chemistry, Cornell Umverslty Press, Ithaca, New York, 1953 J ELIEZER AND H J G HAYMAN, J Polymer SCI , 23 (1957) 387 W BURCHARD, Z Physlk Chem, 42 (1964) 193 M KURATA AND W H STOCK~~AYER, Fortschr Hochpollmer Forsch , 3 (1963) 196 H KUHN, W KUHN, AND A SILBERBERG,J Polymer SCI , 14 (1954) 193 R E RUNDLE AND D FRENCH, J Am Chem Sot, 65 (1943) 558. V S R RAO, P R SUNDARARAJAN, C RAMAKFUSHNAN AND G N_ RAMACHANDRAN, III Co&ormatron of Bropolymers. G N RAMACHANDRAN (Ed ), Academic Press, London, 1967, p 731 0 KRATKY AND G POROD, Ret Trau Chrm ,68 (1949) 1106 H BENOIT AND P DOTY, J Phys Chem, 57 (1953) 958 K NAGAI, J Chem Phjs, 34 (1961) 887 W BROWS, ArkrL Kcnn 18 (1961) 227
Carbohyd
Res , 7 11968) 349-356
Research
349
Ekewer PubhshmmgCompany ~terdam Pnnted III Be&nun
THE CONFORMATION OF AMYLOSE IN NEUTRAL, AQUEOUS SALT SOLUTION* W
BANKS
AND
C
T
GREENWOOD
Department of Chenustry, The Umverslty of Edmbtrrgh, Edmburglr 9 (Great Brltam)
(Recewed January 2nd, 1968)
ABSTRACT
Linear amylose has been subfractlonated, and the hydrodynamxc propeties of the fractions exammed From measurements of the hmdmg-vlscoslty number, 1~1,m 0 33~ potassmm chloride, and of the weight-average molecular weight, i@,, by hght scattermg on the acetate, the relation
was found The slgmficance of the exponent m tlus relation and others m the hteratore IS dzscussed Estimations of the “stiffness” of the amylose molecule m aqueous soiutlon have been obtamed for various model systems, I e , the Kuhn statlstlcal length, the Kratky-Porod persistence length, and Nagal’s theory for a mixture of hehcal and randomly coiled segments The results of each md.vldual theory are ambiguous It 1s shown, on the basis of Nagal’s theory, that there IS defimte evidence for the absence of rlgld helical segments m amylose m aqueous solution The temperature coefficient of [?I] for amylose m aqueous solution 1s also discussed INTRODUCl-ION
The conformation of the amylose molecule m aqueous solution IS currently of much interest Although It has been suggested ’ ’ that the polysacchande undergoes a hehx-coil transformation slmllar to that of proteins, our results3 4 are not consistent with this viewpoint In tks paper, we have extended our earher hydrodynamic measurements on amylose subfractions, and have cntlcally analysed our results m terms of recent theoretical treatments which involve hehcal models EXPERIMENTAL
Linear amylose, z e , a sampIe which was completely hydrolysed mto maltose *Part 43 m the senes “Physlcochemlcal Journal, m the press
Studxs on Starches”,
for Part 42, see European Polymer
Carboh)d
Res ,
7 (1968) 349-356
AMYLOSE
355
CONFORMATION
holds down to molecular werghts of at least 1.5 x 10’ Assuming that, at thus level, R = 10 [see equation (IQ], the maximum fractron of D-glucose resrdues which can be accomodated m the hehces [calculated from equatron (II)] IS of the order of 0.4 Thus, one obtams a model whrch IS composed of essentrally equally-long segments (SO-80 monomer units) of hehx and random co11 This 1sphysrcally unreal, since there 1s no reason why the forces which stabrhzed the helix should be absent over such extended lengths In fact, the use of b, = 1.33 A, mvanably leads to a model whtch 1smconsistent with other hydrodynamic evidence We therefore suggest that, although there may be some helical character m amylose, the compact hehcal segments prevrously postulated ’ ’ do not exist, that is, the hehces are very much “looser” than has been suggested, and b, must be greater than 1 33 A The hehcal nature ofthe macromolecule in solution 1s thus not a result of mtramolecular hydrogen bonding, but merely a consequence of the a-D-(1 +4)-hnkages Indirect evidence for rigid, hehcal segments m amylose IS provided by the temperature-dependence of [q] m aqueous potassmm chloride If, as we have stressed, this 1sa &solvent for the polysacchande, [a] should be very sensmve to temperaturet’ But this IS not the case Over a range of 20-30”, there IS vutually no change m [q] Rao and Foster’ have suggested that the increase m temperature has two opposmg and equal effects the increased solvent-power of the water causes the amylose to expand, whilst the helical segments break down, making the amylose less ngrd and therefore able to take up a more compact conformatton This hypothesis implies that an increase m temperature necessarily leads to an increase m solvent-power, and that consequently solvent-solute mteractron 1s favoured at the expense of solutesolute interaction With aqueous solutrons of amylose, we suggest that the situation 1srather more complex In this case, the polymer 1sheld m solution by hydrogen bonding with the solvent, but, at the same time, polymer-polymer mteractlon 1salso dependent on hydrogen bonding As the temperature IS raised, both types of interaction will tend to become less effectrve Again, these tendencres ~111oppose each other, so that It 1s possrble for [qJ to remam unchanged over a fau-ly wide range of temperature This suggestion satlsfactorrly accounts for the solutton behavlour of amylose m aqueous potassmm chloride as r~functron of temperature zorflmtt mtroducmg a helical model We conclude that the view of amylose as a molecule composed of a number of rigid, helical segments has no experimental basis ACKNOWLEDGMENTS
We have been fortunate enough to have worked m assocratron wrth Professor Srr Edmund Hurst, C B E , F R S , for the past 16 years, and wish to express our indebtedness to him for his continued counsel and the keen interest that he has always taken in our work Thrs work was supported, m part, by a Grant from the U S Department of Agriculture, under P L 480 Carbo!zyd Res ,
7 (1968) 349-356
W. BANKS,
356
C. T
GREENWOOD
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
V S R RAO AND 3 F FOSTER, Btopofymers, 1 (1963) 527 J SZEJTLI AND S AUCXXXAT, St&he, 18 (1966) 38 W BAhKS AND C T GREENWOOD, MaXromol Chem ,67 (1963) 49 W BANKS AND C T. GREENWOOD, m Cotlfornmtron oj Btopolymers, G N RAhiAcHwmrwN (Ed ), Acadermc Press, London, 1967, p 739 W BANKS, C T GREENWOOD, AND D J HOURSTON, Trans Faraday Sor ,64 (1968) 363 R S NICGINBOTHAM AND G A MORRISON, Sh&ey Inst Mem , 22 (1948) 148 C T GREENWOOD AND J S M ROBERTSON, .I Chen? Sot , (1954) 3769 W W EVEREI-I-AND J F FOSTER, J Am Chem SOC 81 (1959) 3464 .l M G COWIE, Makromol Chem, 42 (1961) 230 W BURCHARD, Makromol Chem ,64 (1963) 110 P J FLORY, Prmczples of Polymer Chemistry, Cornell Umverslty Press, Ithaca, New York, 1953 J ELIEZER AND H J G HAYMAN, J Polymer SCI , 23 (1957) 387 W BURCHARD, Z Physlk Chem, 42 (1964) 193 M KURATA AND W H STOCK~~AYER, Fortschr Hochpollmer Forsch , 3 (1963) 196 H KUHN, W KUHN, AND A SILBERBERG,J Polymer SCI , 14 (1954) 193 R E RUNDLE AND D FRENCH, J Am Chem Sot, 65 (1943) 558. V S R RAO, P R SUNDARARAJAN, C RAMAKFUSHNAN AND G N_ RAMACHANDRAN, III Co&ormatron of Bropolymers. G N RAMACHANDRAN (Ed ), Academic Press, London, 1967, p 731 0 KRATKY AND G POROD, Ret Trau Chrm ,68 (1949) 1106 H BENOIT AND P DOTY, J Phys Chem, 57 (1953) 958 K NAGAI, J Chem Phjs, 34 (1961) 887 W BROWS, ArkrL Kcnn 18 (1961) 227
Carbohyd
Res , 7 11968) 349-356