. d2. Chem. Papers 44(\)\\\ 117 (1990)

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1 Dstrbuton pattern of 4OmethylDglucuronc acd unts n 4OmethylDglucuronoDxylan solated from the leaves of marsh mallow {Althaea offcnals L., var. Rhobusta) A. KARDOŠOVÁ, A. MALOVÍKOVÁ, J. ROSÍK, and P. CAPEK Insttute of Chemstry, Centre for Chemcal Research, Slovak Academy of Scences, CS84238 Bratslava Receved 9 February 1989 Dedcated to Professor P. Krstán, DrSc, n honour of hs 60th brthday A homogeneous, watersoluble 4OmethylDglucuronoDxylan has been solated from the leaves of marsh mallow. The mole rato of Dxylose and 4OmethylDglucuronc acd was 7:1. The man chan was bult of (1 4)lnked ßDxylopyranosyl resdues and 4OmethylDglucuronc acd was attached as sngle termnal unts to 02 of Dxylose. The sngleon actvty coeffcent of calcum counterons y c. d 2 f was estmated n a molecular dsperse soluton of the polysaccharde. The mean dstance of adjacent carboxyl groups, b = 4.00 nm, was obtaned from expermentally determned value of / Ca2+ = 0.712, usng the relaton Xca2+ =f(b). From comparson of the b value wth the length of the Dxylose unt (0.52 nm) t follows that the uronc acd unts are apart and dstrbuted regularly along the macromolecule chan,.e. approxmately each seventh Dxylose unt s substtuted by 4OmethylDglucuronc acd. Из листьев алтея аптечного выделен гомогенный водорастворимый 40метилоглюкуронооксилан. Мольное отношение DКСИЛОЗЫ И 40метилоглюкуроновой кислоты в нем было 7:1. Главная цепь состояла из (1^связанных остатков ßDксилопиранозы, причем 4Ометилоглюкуроновая кислота была присоединена в виде одиночных концевых групп к атому 02 DКСИЛОЗЫ. Величина одноионного коэффициента активности кальциевых противоионов / Са 2+ была определена в молекулярно дисперсном растворе полисахарида. Значение среднего расстояния между соседними карбоксильными группами, b = 4,00 нм, было получено на основе опытно установленного значения y c. d2 + = 0,712, используя соотношение / Са 2+ =f(b). Из сравнения величины b с размером единицы DКСИЛОЗЫ (0,52 нм) следует, что единицы уроновой кислоты не находятся рядом, а регулярно распределены вдоль цепи макромолекулы, т.е. приблизительно каждая седьмая единица DКСИЛОЗЫ замещена 40метилоглюкуроновой кислотой. Elucdaton of sequental arrangement of 4OmethylDglucuronc acd n the Dxylan molecule s not an easy task. Partal acd or enzyme hydrolyss Chem. Papers 44(\)\\\ 117 (1990) 111

2 A. KARDOŠOVÁ. A. MALOVÍKOVÁ, J. ROSÍK. P. CAPĽK followed by fractonaton and characterzaton of olgomerc fragments s tme consumng and may lead to ambguous conclusons. Earler [1] t was generally accepted that n glucuronoxylans the monomerc uronc acd unts are dstrbuted randomly along the Dxylan chan. However, Shmzu and coworkers [2] solated a tetramer contanng two vcnal unts of 4OmethylDglucuronc acd bound to 02 of Dxylobose from the hydrolyzates of hemcelluloses of neutral sulfte waste lquor of larch. Lately, Kohn et al. [3,4] have suggested a blockwse dstrbuton of 4OmethylDglucuronc acd n 4OmethylDglucuronoD xylans solated from the bark of whte wllow and beech. They came to ths concluson on the bass of the results obtaned by a novel methodc approach,.e. by determnaton of the sngleon actvty coeffcent of calcum counterons (ľca 2 +) m solutons of calcum salts of these polysacchardes. In the present work we made use of ths method n determnaton of dstrbuton pattern of sde 4OmethylDglucuronc acd unts n 4OmethylD glucuronodxylan solated from the leaves of marsh mallow (Althaea offcnals L., var. Rhobusta). Expermental Potentometre ttratons were performed on a PHM 64 (Radometer, Copenhagen) potentometer usng a G К 2401 С combned electrode. Spectrophotometry measurements were carred out on a Specol 11 (Zess, Jena) spectrometer. The Cd spectra of both potassum and calcum salts of the polysaccharde were measured n 1.00 mmol dm" 3 solutons of COOK and COOCa () 5 wth a Jobn Yvon Mark III (France) dchrograph. Reagents used: 0.058MKOH, MCa(OH) 2, MCaCl 2, redstlled water freed from carbon doxde, and tetramethylmurexde syntheszed accordng to the method descrbed n [5]. Isolaton and characterzaton of the polysaccharde The solaton of 40methylDglucuronoDxylan from the leaves of marsh mallow and ts subsequent separaton from the accompanyng polysacchardes were carred out as descrbed n our prevous work [6]. The structural features, determned by methylaton analyss and l3 C NMR spectroscopy, are descrbed n the same work. The content of 4OmethylDglucuronc acd was determned by Potentometre ttraton and the methoxyl content accordng to the method of Vebock and Brecher [7]. Both methods afforded consstent results. The number average molecular mass (M n ) was determned osmometrcally at 30 C after equlbraton wth 0.1 MNaCl, usng a Knauer membrane osmometer ftted wth a ZweschchtMembrane (Knauer). 112 Chcm. Paper* 44 (1) (1990)

3 40METHYLDGLUCURONODXYLAN Determnaton of actvty of Ca 2+ ons n the soluton of calcum salt of 40methylDglucuronoDxylan The polysaccharde was converted nto H + form by percolatng the polysaccharde soluton through Amberlte IR 120 (H + ) and then t was neutralzed to the pont of equvalence wth saturated Ca(OH) 2 soluton. The actvty of Ca 2+ ons was determned n the soluton of calcum salt of the polysaccharde (<ľ(cooca 05 ) = 3.00 mmol dm ~' 3 ) by the spectrophotometrc method usng tetramethylmurexde (c = 4x 10 _:> moldm~ 3 ) as the metallochromc ndcator [8, 9]; the soluton dd not contan any addtonal nert electrolyte. The calbraton curve was constructed from the data obtaned wth CaCl 2 soluton. The sngleon actvty coeffcent y Ca 2+ was calculated from the Ca 24 actvty and the total concentraton of Ca 24 n the soluton (1.5 mmol dm" 3 ). Determnaton of dstrbuton of 4OmethylDglucuronc acd n the polysaccharde The mean dstance (b/nm) between two adjacent free carboxyl groups n ther perpendcular projecton on the axs of the Dxylan lnear chan was estmated from the analytcal curve y Cl2 + =f(b). Ths functon was ntroduced n our earler paper [10] usng / Ca 2+ determned n model solutons of varously esterfed pectns (E > 43 %) [11]. Results and dscusson A homogeneous, watersoluble 4OmethylDglucuronoDxylan has been solated from the leaves of marsh mallow. The polysaccharde was composed of (l»4)lnked /?Dxylopyranosyl resdues, ^70% beng unsubsttuted, «5% carryng a sngle substtuton at 02 and/or 03, and ^11 % beng doubly branched on 02 and MethylDglucuronc acd was attached to 02 of Dxylose as sngle termnal unts. The mole rato of Dxylose to 40methylD glucuronc acd n the polysaccharde was 7:1 (Table 1). Table 1 Characterzaton of 4OmethylDglucuronoDxylan w (DXylose) w (4OMethylDglucuronc acd) vr(och0 M n [Ö](D, 22 C, Q= 5.0gdm~ 3, water) Mole rato Dxylose: 4OmethylDglucuronc acd 83.10% 16.90% 2.72 % :1.00 Chem. Papers 44 (1) (1990) 113

4 A. KARDOŠOVÁ, A. MALOVÍKOVÁ, J. ROSÍK, P. CAPEK The prncple of the method for determnaton of the dstrbuton pattern of carboxyl groups n an acd polysaccharde molecule, based upon nterpretaton of sngleon actvty coeffcent of calcum counterons (y Ca 2+), has already been descrbed [10]. The prerequste for applyng the foregong method s the electrostatc bond between the calcum ons and carboxyl groups of the polysaccharde. The actvty coeffcent of counterons (yx bound by electrostatc bond to the polysaccharde, s a functon of lnearcharge densty of the macromolecule characterzed by the mean dstance between two adjacent carboxyl groups (6/nm). The functon y Ca2+ =f{b) (Fg. 1) has a general valdty for lnear acd Fg. 1. Dependence of / Ca2+ on the mean dstance (b) of two vcnal free carboxyl groups n a lnear polysaccharde. polysacchardes, regardless of the knd of uronc acd unts n the polysaccharde. As t was mentoned n Expermental, Fg. 1 was constructed from the values of actvty coeffcents determned n solutons of calcum salts of pectns havng more than 43 % carboxyl groups esterfed, wheren the bond between calcum and carboxyl groups was exclusvely electrostatc [11]. The dentty of crcular dchroc spectra (Fg. 2) of calcum and potassum salts of the glucuronoxylan studed heren ndcated that the bond between calcum and carboxyl groups was electrostatc [12]. The spectra, recorded wth transparent solutons (concentraton of COOK and COOCa^ = 1.00 mmol dm" 3 ), revealed the maxmum n the negatve value of the ellptcty range at A = 210nm. The actvty of Ca 2+ counterons (tf C a 2 +) was estmated n the soluton of calcum salt of 40methylDglucuronoDxylan (c(coocao 5 ) = 3 mmol dm 3 ) and the actvty coeffcent y c. d + =0.712 was calculated. Ths value s very 114 Chem. Papers 44(\)\\\ 117(1990)

5 40METHYLDGLUCURONODXYLAN smlar to that found at the same concentraton wth calcum Dglucuronate ( 7ca2+ = 0.755). The dstance between two vcnal carboxyl groups n the polysaccharde At 0 2 1\ Са У к Л/nm Fg. 2. Crcular dchroc spectra of potassum and calcum salts of 40methylDglucuronoDxylan (concentraton of COOK and COOCa 05 = 1 mmol dm ~ 3 ). У Са 2 * I 1 4 " 3 So Ь7пт 1 Fg. 3. Lnearzed form (\/у Са 2+ =/(!//?)) of the relatonshp llustrated n Fg. I. Chem. Papers 44(\)U\ 117(1990) 115

6 A. KARDOŠOVÁ, A. MALOVÍKOVÁ, J. ROSÍK, P. CAPEK b = 4.00 nm, correspondng to the found actvty coeffcent, was obtaned from the lnearzed form (1// C a 2 + =f0/b); Fg. 3) of the functon y Ca2+ =f(b) (Fg. 1). Though ths relatonshp was proved to be vald for lnear acd polysacchardes [11],.e. when the uronc acd unts are bult n the lnear chan, we consder ths dependence applcable to our polysaccharde, snce 40methylDglucuronc acd s attached to the man lnear chan exclusvely as sngle termnal unts. When consderng the analogy wth the macromolecule of Dmannuronan, havng the same type of glycosdc bonds (ß(\ >4)) of the saccharde unts n 4 C, conformaton, the length of one Dxylose unt n the Dxylan chan s 0.52 nm [13]. Then from the b value 4.00 nm t follows that approxmately each seventh Dxylose unt n the macromolecule s substtuted by one unt of 4OmethylD glucuronc acd. Ths concluson s n agreement wth the results of structural studes. Table 2 Mean dstance between two adjacent carboxyl groups (b) n 40methylDglucuronoDxylans n a perpendcular projecton on the man axs of ther molecules Source Mole rato v T A Ĺ / Xyl: UA b/nm Bark of whte wllow [3] Beech wood [4] Leaves of marsh mallow For comparson, we present n Table 2 the b values determned wth 40 methyldglucuronodxylans solated from the bark of whte wllow and beech wood [3, 4]. These pont to rregular dstrbuton of uronc acd unts n the macromolecule. The polysacchardes are composed of blocks wth more frequent branchng (approxmately at each second xylose unt), alternatng wth those composed exclusvely of xylose unts or contanng only small amount of uronc acd unts. Contrary to these polysacchardes, n the 40methylD glucuronodxylan studed heren, the uronc acd unts are apart and dstrbuted regularly along the macromolecule chan. Ths fndng completes the knowledge on the structure of ths polysaccharde. Moreover, t may be of mportance n vew of nvestgatons and possble classfcatons of further types of glucuronoxylans or hemcelluloses n general, consderng ther orgn. Acknowledgements. The authors thank M. Bys t ran for techncal assstance. 116 Chem. Papers 44(\)\\\ 117(1990)

7 40MF,THYLDGLUCURONODXYLAN References 1. Tmell, T. E., Adv. Carbohydr. Chem. 19, 247 (1964). 2. Shmzu, K. and Samuelson, O., Svensk Papperstdn. 76, 156 (1973). 3. Toman, R., Kohn, R., Malovíková, A., and Rosík, J., Collect. Czechoslov. Chem. Commun. 46, 1405(1981). 4. Kohn, R., Hromádková, Z., Ebrngerová, A., and Toman, R., Collect. Czechoslov. Chem. Commun. 51, 2243 (1986). 5. Gyslng, H. and Schwarzenbach, G., Helv. Chm. Acta 32, 1484 (1949). 6. Kardošová, A., Capek, P., and Rosík, J., Chem. Papers (1989). 7. Vebock, F. and Brecher, C, Ber. 63В, 3207 (1930). 8. Kohn, R. and Furda, I., Collect. Czechoslov. Chem. Commun. 32, 1925 (1967). 9. Kohn, R., Chem. Zvest 28, 625 (1974). 10. Kohn, R., Rosík, J., Kubala, J., and Malovíková. A., Collect. Czechoslov. Chem. Commun. 44, 2517(1979). 11. Kohn, R. and Luknár, О., Collect. Czechoslov. Chem. Commun. 40, 959 (1975). 12. Kohn, R. and Stczay, Т., Collect. Czechoslov. Chem. Commun. 42, 2372 (1977). 13. Atkns, E. D. Т., Pure Appl. Chem. 49, 1135 (1977). Translated by A. Kardošová Chem. Papers 44 (1) (1990) 117