Journal of Chromatography,
403 (1987) 373-378
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. 19 638
Separation of amylose
of starch components on cellulose
based on ligand-induced
YASMIN Y. TALIB, MEENA S. KARVE, SHOBHANA V. BHIDE and N. R. KALE* Department of Chemistry, Division
qf Biochemistry, University of Poona, Pune 411 007 (India)
(First received August 27th, 1986; revised manuscript received March 3Oth, 1987)
The polysaccharides deposited in starch granules normally consist of an heterogeneous mixture of linear (amylose, 20-25%) and branched (amylopectin, 7580%) isotactic homopolymers of D-glucose. Amylose is made up 01 linear chains of D-glucopyranose units linked through a-( 1 -+ 4) bonds and is heterogeneous with respect to the degree of polymerization (1 . 102-1 . 104). Amylopectin contains short linear chains of a-(1 + 4) linked D-glucopyranose residues f17-23), which are interconnected mainly by ~(1 + 6) linkages to form an highly branched structure. It is heterogeneous with respect to the degree of branching and the degree of polymerization (1 . 104-1 . 105). Amylose and amylopectin are held together by hydrogen bonds, either directly or through water molecules. Most of the starches contain an intermediate fraction (4-9%)’ which is characterized as amylose with a few a-(1 + 6) linkages that are resistant to the action of P-amylase but hydrolysed by pullulanase2, Starches from certain sources such as amylomaize or wrinkled pea are rich in amylose (66~80%), whereas waxy maize or waxy sorghum contains no amylose, as judged by their iodine-binding capacity. Chromatographic procedures for the separation of starch components are mostly based upon differences in solubility and the preferential adsorption of one of the components or its complex with a suitable ligand. Amylose is known to form an helical complex with a variety of ligandsl such as I-butanol, 1-pentanol, cyclohexanol, thymol, sodium dodecyl sulphate (SDS), iodine, etc. The chromatographic separation of starch components on filter-paper strips using 40% perchloric acid as an irrigant has been reported by Taki3 and Richter and Stroh4: According to Taki5, starch dispersed in perchloric acid is developed in the same acid in the presence of iodine vapours at 5°C. The amylopectin-iodine complex migrates but the amyloseiodine complex remains at the point of application. In the absence of iodine, the amylopectin does not move and the amylose migrates accompanied by a ,small amount of amylopectin. These procedures are tedious and the use of strong acid is bound to cause extensive degradation of starch components. The separation of starch components on filter-paper can also be obtained by using 0.2-1.0 M potassium hydroxide6g7, as an irrigant. The amylopectin remains at the point of application (RF = 0), while amylose moves with the solvent (RF = 0.5). Both amylose and amylopectin are prone to oxidative degradation in alkaline solution in the presence of oxygen, though the reaction is slow at room temperature. These procedures can 0021-9673/87/$03.50
1987 Elsevier Science Publishers B.V.
be useful for determining the amylose and amylopectin contents of different starches as well as the purity of starch components obtained by precipitation using different ligands3. Attempts have been made to separate starch components using different gel filtration media. The elution profiles of starch components on different gel filtration media such as Sephadex G-2008, Sepharose 2Bs$g, Toyopearl HW-7.5Fl”, Sephacryl S-10001 l, have shown that amylopectin appears in the void volume and amylose, depending on the molecular weight, enters the gel matrix. Recently ,Kobayashi et al. l2 reported an high-performance size-exclusion chromatographic (HPSEC) procedure for rapid separation (20 min) of starch components. Earlier we reported the separation of starch components by affinity chromatography13 and adsorption on cellulose14. Here we describe a simple filter-paper chromatographic method for the separation of starch components on a micro-scale (250-500 pg), based on the ligandinduced adsorption of amylose on cellulose. MATERIALS
Potato starch was prepared according to the method of Schochr5. The corn, tapioca and sorghum starches were commercial products obtained from a local market. The starch granules were defatted by repeated extraction with chloroforn-methanol (2:1, v/v) at 80°C. Preparation of starch solution The defatted starch granules were dispersed in 1.0 M sodium hydroxide at room temperature (2628°C) by vortexing in an atmosphere of nitrogen, followed by neutralization with 1.0 M hydrochloric acid. The freshly prepared starch solution (I%, w/v) was clarified by centrifugation at 12000 g and used for spotting. This procedure minimizes the alkaline degradation of starch components during dispersion. n-Amylase (E.C. 22.214.171.124) from human saliva was prepared according to the method of Bernfeld16. P-Amylase (E.C. 126.96.36.199) from sweet potato was prepared according to the method of Balls et al. 17, and Nakayama and Anagasel 8. Pullulanase (E.C. 188.8.131.52) was obtained from Nakarai Chemicals Ltd. (Japan). Solvent system A 0.1 A4 acetate buffer pH 4.8 containing 2 A4 urea was saturated with ligands such as 1-butanol, l:pentanol, cyclohexanol at room temperature (2628°C) for 16-18 h. The aqueous phase saturated with the respective ligands was used. Solutions of ethanol (32%, v/v), thymol(O.l3%, w/v) and 1 . lop4 M sodium dodecyl sulphate (SDS) were used. The iodine solution contained 7.87 . 1O-4 M iodine and 1.21 . low2 M potassium iodide in distilled water. Filter-paper (Whatman No. 3) strips (40.0 cm x 5.0 cm) were rinsed with an alkaline solution of 120 mA4 sodium carbonate pH 8.6 containing 1 mM EDTA, followed by repeated washings with distilled water and drying in air. They were wetted at the point of application with the solvent system, excess of the solvent was removed and the sample of dispersed starch (250-500 yg; lo!, w/v) was applied with an automatic micro-pipetter. The paper was developed with a suitable solvent system
in the descending direction, in a thermostated chromatographic chamber (30 f 05°C) for 4-5 h. Two beakers containing the aqueous phase and the organic phase respectively were placed at the bottom of the chromatographic chamber. The filterpaper strips were washed with ethanol (95%, v/v) to remove the urea and to fix the separated starch components. They were then dried in air and sprayed with iodine solution (KI;) to visualize the spots of amylose and amylopectin. The amylose spot at the point of application gave a blue colour due to formation of the amylosetriiodide complex, while the amylopectin spot near the advancing front gave a purple colour due to the amylopectintriiodide complex. The spots containing the starch components were eluted with 0.1 h4 acetate buffer pH 4.8 containing 2 A4 urea and centrifuged at 12000 g. The clear solution devoid of cellulose fibres was used for the estimation of polysaccharide by the phenol-sulpohuric acid method1 g. For enzymatic assay, the spots were removed, incubated in 0.1 M phosphate buffer pH 6.9 containing 100 units of a-amylase or in 0.1 M acetate buffer pH 4.8 containing 100 units of P-amylase at 30°C for 8 h, and the maltose liberated was determined by Nelson’s methodzO. The blue value (absorbance of the polysaccharide at 680 nm in the presence of KIT) of the extracted polysaccharide samples was determined by the method of McCready and Hassid2 l. For blank experiments, pieces of filter-paper of equal size were processed under identical conditions. RESULTS AND DISCUSSION
Our earlier studies14 had shown that starch (2%, w/v).dispersed in 2 A4 urea forms as clear solution which is stable at room temperature (2628°C) for several months. Hence we have used 0.1 M acetate buffer pH 4.8 containing 2 M urea, which also helps to prevent the formation of ammonium cyanate. According to Erlander and Tobinz2, urea helps to stabilize the helical conformation of amylose in solution. In the chromatographic separation of starch components on filter-paper, using different solvent systems, we observed that the amylose-ligand complex remains at the point of application, while the amylopectin-ligand complex moves along with the solvent front. The spots have tails, indicating the heterogeneous nature of the starch components (Fig. 1A). The amylose spot was cut into three equal parts (Fig. lB), eluted with 2 M urea and the polysaccharide content was determined by the phenol-sulphuric acid method19. The amylose was further characterized by determining the blue value and the P-amylolysis limit. The results (Table I) indicate that potato amylose consists of an heterogeneous population of amylose chains which differ in their branching. The amylose at the point of application (I) appears to be a high-molecular-weight fraction, fraction II is an intermediate fraction and fraction III has a low degree of branching, as revealed by their blue values and P-amylolysis limits. The amylose from fractions II and III on treatment with pullulanase gave a b-amylolysis limit of 98% implying the presence of few a-(1 --+ 6) linkages. The method is very simple and permits a clear-cut separation of linear (amylose) and branched (amylopectin) components of defatted starch in a short time. The high recovery (90-95%) of the polysaccharide suggests that this method can be used for quantitative separation and estimation of starch components on a microscale (25G-500 pg) during the development of seeds. Our results on the quantitation of
Fig. 1. Chromatographic separation of starch (potato) components on filter-paper (Whatman 0.1 A4 acetate buffer pH 4.8’ containing 2 M urea and I-butanol. . = Point of application; solvent front: 1 = amylose: 2 = amylopectin. See text for further details.
No. 3) using -----=
starch components from different starches have shown that this method gives reproducible results with good accuracy (amylose, 65.0 f 2.5 pg; amylopecfin, 180 f 8.0 pg and starch, 250 f 9.5 ,ug). The values mentioned are mean values f standard deviations obtained from seven replicate experiments. We have analysed the amylose and amylopectin contents of different starches (Table II). The amylopectin from corn starch gave two spots, which differ in their blue values and j?-amylolysis limits. In the case of degraded starch like soluble starch (potato), amylose and amylopectin gave highly tailed spots. We have studied the separation of potato starch components using different ligands such as ethanol; I-pentanol, cyclohexanol, 1-butanol, thymol (0.13%, w/v) and 1.0 . lop4 M SDS in 0.1 M acetate buffer pH 4.8 containing 2 M urea. All these TABLE
I II III
Polysaccharide (%) !
NOTES TABLE II CHARACTERIZATION
OF STARCH COMPONENTS
A = Amylose; B = amylopectin.
Starch Potato Corn
Soluble (Potato) Sorghum Tapioca Rice
A B A B (I) (II) A B A B A B A B
/I-Arnylolysis IimiP W)
1.35 0.18 1.35 0.25 0.18 1.30 024.22 1.35 0.18 1.35 0.18 1.35 0.18
9&95 48-50 9&95 5tio 48-50 9G95 6&62 9&95 48-50 90-95 48-50 90-95 48-50
ligands gave good separations of potato starch components. In case of the SDS system it was very difficult to remove traces of SDS from amylose and amylopectin fractions even after repeated extraction with hot methanol The SDS interfers in the determination of the blue value, but not the P-amylolysis limit. If the concentration of urea is increased to 3 or 4 M in 1-butanol in 0.1 A4 acetate buffer pH 4.8, the spot due to the amylose-butanol complex moves from the point of application, along with the amylopectin-butanol complex thus the separation is not clear cut. It is interesting that amylose is not adsorbed on filter-paper, but the amylose-ligand complex has an high affinity for cellulose. The interaction of amylose with certain ligands23*24 results in a coil -+ helix transition with ligand molecules entrapped in the helical lumen, resulting in the formation of some hydrophobic domains on the surface of the helical complex. These domains probably interact with amorphous domains of cellulose 25 to immobilize the amylose-ligand complex. We have shown that crystalline cellulose obtained by treating cellulose powder with dilute acid has no affinity for the amylose-ligand complex. ACKNOWLEDGEMENT
Financial assistance from the Science and Technology Cell, Education partment, Government of Maharashtra, Bombay is-gratefully acknowledged.
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