Tobacco pectin

Structural Characterization and Visualization In Situ and After Isolation of Tobacco Pectin... 

Recently two different disciplines, chemical structural elucidation and transmission electron microscopy, were utilized in the study of pectin, with particular emphasis on tobacco pectin. The goal was to help bridge the gap between knowledge of their chemical structures to understanding the complex physical structures revealed by microscopy. To provide background on chemical structure, a study established that tobacco pectin was present as a series of related rhamnogalacturonans. 

Pectins are best known for their ability to form gels (6), a property which often involves intermolecular binding mediated by calcium cations (7). The principal commercial use of pectin is in the preparation of jelly and jam products (8). Pectins provide firmness in fresh fruits and vegetables (9-11). Historically, natural tobacco pectins also have been used as binders to prepare reconstituted sheets from tobacco by-products that are then incorporated into cigarette filler or cigar wrappers (12-14). 

The results from structural studies on pectins isolated from a number of different plant sources have been reported in several papers and review articles (10,22-28). Chemical investigations of tobacco pectin (15,29-31) have demonstrated that its structure is consistent with the basic structural elements found in pectins from other sources. 

In one recent study (15), isolation and purification of tobacco pectin yielded a series of related rhamnogalacturonans. All of these polysaccharides were found to have a backbone consisting of 4-linked a-D-galactopyranosyluronic acid residues interspersed with 2-linked L-rhamno-pyranosyl residues in a ratio of 16 1 (see Fig. 1). The presence of rhamnose in the backbone of pectin is believed to create kinks which probably disrupt helical stretches of the 4-linked a-D-galactopyranosyluronic acid... 

Figure 1. Model chemical structure of tobacco pectin.

Gels were obtained in the following manner from both the purified, starting tobacco pectin and the deesterified pectin obtained from it. First the sample of pectin was solubilized in deionized 100°C water ( 1% solution). Then the pectin was gelled by ethanol vapor, introduced slowly (6 hrs) by surrounding the vessel containing aqueous pectin with 100% ethanol in a closed container at 20° C. 

The gel from the purified tobacco pectin was formed on 1.3 cm ashless Whatman 50 filter paper discs which were frozen in propane at about — 190°C. These samples were then freeze-dried (90 min) at —70°C, replicated with 16.9A Pt/C at —178°C in a 5 x 10-8 torr. vacuum and backed with 146A of carbon. Finally, these samples were digested with 80% sulfuric acid, rinsed with deionized water, picked up from underneath with... 

Figure 4A shows a relatively low magnification micrograph of a gel prepared from deesterified tobacco pectin. Unlike Figures 2A and 3A, its surface was smoother and did not show the layering effect seen on the lower epidermal cell surface. 

Since the x-ray fiber diffraction measurements based on citrus pectin (32) were consistent with the TEM measurements of tobacco pectin, we prepared a gel from citrus pectin similar to the previous x-ray sample. This gel was then examined by TEM. Air-dried samples of this gel, shown in Figure 5, demonstrated long stretches of helix in the molecules lying on the surface. (In the freeze-dried gels—not shown—only short stretches of helix were visible.) The average filament width in the air-dried gel was found to be 14.2A. After correcting for the added size due to the Pt/C coating... 

In conclusion, we have demonstrated that high resolution TEM is a valuable complement to x-ray fiber diffraction analysis and chemical structural elucidation. Its application provided information about the organization of pectin in cell walls and in calcium-free gels. Using freeze-dried samples that were Pt/C replicated, we demonstrated tobacco pectin filaments in a gel to be of the same diameter as the filaments on the noncutinized lower epidermal surface of senescing Coker 319 tobacco leaves. These filaments were 7.1 3A and 4.6 4.8A, respectively, and roughly the same diameter, 7A, as fiber-diffraction modeled citrus pectin (32). Replicated... 

Figure 4A. Freeze-dried, Pt/C replicated gel prepared from deesterified tobacco pectin. (Bar = 10,000A.)...

We believe that single molecule imaging can contribute to a more thorough understanding of the role of pectin in the cell wall. Our future efforts will be focused on pectin gels formed in the presence of calcium. Eventually, it should be possible to visualize side chains on pectin and determine how rhamnose residues in the rhamnogalacturonan backbone of tobacco pectin disrupt the formation of helical regions. 

Pyriki, C. and W. Moldenhauer Investigations on tobacco pectins and their influence on the quality of tobacco Ber. Inst. Tabakforsch. Dresden 10 (1963) 238-263 Tobacco pectins and their influence on the quality of tobacco Nahrung 7 (1963) 539-544. 

Young, H.J. Reconstituted sheet made from cigarette dust. The use of tobacco pectin for production of reconstituted tobacco products R DM, 1988, No. 280,... 

Tobacco sheets, made from t. dust, and binders based on tobacco pectins or ->-methylcelIulose, have to contain at least 75% tobacco and are used in cigar rolling ... 

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