Problems encountered during the extraction, purification, and chromatography of pectic fragments, and some solutions to them

Mort, Andrew J.; Moerschbacher, Bruno M.; Pierce, Margaret L.; Maness, Niels O.

doi:10.1016/0008-6215(91)84022-7

Cited by 121 publications

(80 citation statements)

References 19 publications

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“…The suspensions were filtered through GF/C filters, and the filtrates were measured for soluble uronic acids (Blumenkrantz and Asboe-Hansen, 1973). As an alternative to the use of CDTA, 500 mM imidazole, pH 7.0, was also used to extract chelator-soluble polyuronides (Mort et al, 1991) from AIS. In some experiments, AIS following extraction in CDTA-containing buffers were transferred to Na2C03 and maintained for 6 h at 4OC (Jarvis, 1982).…”

Section: Extraction Of Chelator-soluble Polyuronidesmentioning

confidence: 99%

“…Similar buffer conditions have been shown to minimize intermolecular aggregation and to yield more uniform behavior of polyuronides on gel filtration matrices (Mort et al, 1991). Two-milliliter fractions were collected at a flow rate of 15 mL h-I, and 0.5-mL aliquots were assayed for uronic acid content.…”

Section: Cel Chromatography Of Polyuronidesmentioning

confidence: 99%

“…Polyuronide solubility was also examined using 500 mM imidazole, pH 7.0 (Mort et al, 1991). As summarized in Table I Sepharose CL-2B-300 profiles of CDTA-soluble polyuronides derived from cv Hass avocado mesocarp tissue.…”

Section: Uronic Acid Content and Solubility In Ais Derived From Avocamentioning

confidence: 99%

“…The higher M, values observed in the present study is likely explained by the use of protocols designed to achieve more rapid inactivation and denaturation of tissue enzymes. Chromatography conditions used (ionic strength, pH) were based on those reported to minimize intermolecular aggregates and to yield highly reproducible elution behavior (Fishman et al, 1989;Mort et al, 1991).…”

Section: M Analysis Of Cdta-soluble Polyuronides From Avocado Fruit mentioning

confidence: 99%

“…These observations indicate that the environment provided by the release of endogenous cellular fluids is favorable for enhancing polyuronide depolymerization and, therefore, accentuating differences in the PG antisense versus normal tomato lines. In addition to stimulating enzymic hydrolysis of polyuronides, altered ionic strength could have the effect of causing nonenzymic dissociation of noncovalently associated polyuronide aggregates into smaller subunits (Fishman et al, 1989;Mort et al, 1991).…”

Section: Possible Role Of Polyuronide Depolymerization and Pc In Ripementioning

confidence: 99%

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Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening

1993

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Avocado (Persea americana) fruit experience a rapid and extensive loss of firmness during ripening. In this study, we examined whether the chelator solubility and molecular weight of avocado polyuronides paralleled the accumulation of polygalacturonase (PC) activity and loss in fruit firmness. Polyuronides were derived from ethanolic precipitates of avocado mesocarp prepared using a procedure to rapidly inactivate endogenous enzymes. During ripening, chelator (cyclohexane-trans-l,2-diamine tetraacetic acid [CDlA])-soluble polyuronides increased from approximately 30 to 40 pg of galacturonic acid equivalents (mg alcohol-insoluble solids)-' in preripe fruit to 150 to 170 pg mg-' in postclimacteric fruit. In preripe fruit, chelator-extractable polyuronides were of high molecular weight and were partially excluded from Sepharose CL-26-300 gel filtration media. Avocado polyuronides exhibited marked downshifts in molecular weight during ripening. At the postclimacteric stage, nearly all chelator-extractable polyuronides, which constituted from 75 to 90% of total cell wall uronic acid content, eluted near the total volume of the filtration media. Rechromatography of low molecular weight polyuronides on BioCel P-4 disclosed that oligomeric uronic acids are produced in vivo during avocado ripening. l h e gel filtration behavior and pattern of depolymerization of avocado polyuronides were not influenced by the polyuronide extraction protocol (imidazole versus CDTA) or by chromatographic conditions designed to minimize interpolymeric aggregation. Polyuronides from ripening tomato (fycopersicon esculentum) fruit extracted and chromatographed under conditions identical with those used for avocado polyuronides exhibited markedly less rapid and less extensive downshifts in molecular weight during the transition from mature-green to fully ripe. Even during a 9-d period beyond the fully ripe stage, tomato fruit polyuronides exhibited limited additional depolymerization and did not include oligomeric species. A comparison of the data for the avocado and tomato fruit indicates that downshifts in polyuronide molecular weight are a prominent feature of avocado ripening and may also explain why molecular down-regulation of PC (EC 3.2.1.15) in tomato fruit has resulted in minimal effects on fruit performance until the terminal stages of ripening.Avocado fruit soften extensively during ripening, with mesocarp firmness, measured in terms of resistance to penetration, exceeding 450 N at the preripe stage and decreasing severa1 orders of magnitude during ripening (Pesis et al., 1978; Awad and Young, 1979; O'Donoghue and Huber,

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Section: Extraction Of Chelator-soluble Polyuronidesmentioning

confidence: 99%

Section: Cel Chromatography Of Polyuronidesmentioning

confidence: 99%

Section: Uronic Acid Content and Solubility In Ais Derived From Avocamentioning

confidence: 99%

Section: M Analysis Of Cdta-soluble Polyuronides From Avocado Fruit mentioning

confidence: 99%

Section: Possible Role Of Polyuronide Depolymerization and Pc In Ripementioning

confidence: 99%

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Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening

1993

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Extraction of pectic polysaccharides from sugar-beet cell walls

Marry

McCann

Kolpak

et al. 2000

J. Sci. Food Agric.

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Previous methods of extracting pectin from sugar-beet have used pulp as the starting material. As the temperature and pressure of the pulping process may modify the architecture of the cell wall, we have adapted a relatively non-disruptive method to characterise cell wall material (CWM) isolated directly from the sugar-beet. Cell walls from mature sugar-beets (Beta vulgaris L Aztec) were sequentially extracted four times with imidazole and twice with sodium carbonate to produce six heterogeneous pectic polysaccharide extracts, and with KOH to produce a hemicellulosic extract which was predominantly xylans. Heterogeneity of the extracted pectins was indicated by differences in FTIR spectra, uronic acid content, % methyl esteri®cation, % feruloylation, % acetylation, molecular weight distribution and neutral sugar composition. The highest proportion of feruloyl esters was found in polysaccharides solubilised by the second sodium carbonate extraction. Anion exchange chromatography of these polysaccharides gave three fractions, one of which contained most of the feruloyl ester. These results indicate that feruloyl esters are not randomly distributed among the different pectic polysaccharides in the sugar-beet cell wall, and that esteri®cation is likely to be dependent on the local sugar sequence or conformation.

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Isolation of Plant Cell Walls and Fractionation of Cell Wall Polysaccharides

Melton

Smith

2001

Current Protocols in Food Analytical Chemistry

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Plant foods are an important component of the diet of humans. Since the cell wall makes up a large component of the dry weight of plant tissues, and because in a normal diet most of the dietary fiber comes from plant cell walls, it follows that there is a need to know the composition of the cell walls. Moreover, the cell wall contributes (along with turgor pressure and the cell contents) to the texture of plant foods, and since humans consider the texture of foods an important sensory attribute, again, understanding wall composition is important. While for some plant species the cell wall composition has been studied, the actual three-dimensional organization of the polysaccharides in the cell wall has not been fully elucidated. Moreover, it appears that while some aspects are constant for all walls (i.e., the presence of similar types of polysaccharides), the relative proportion and fine structure of the polysaccharides may be different, and thus the way they are organized in the wall may be different. Determination of the composition of polysaccharides in cell walls is commonly done by chemical analytical methods, but prior to determining the composition or structure of the polysaccharides in cell walls, the walls must first be isolated from the plant tissues and separated from the intracellular material. The first step in isolating cell walls is to examine the plant material microscopically (see Basic Protocol 1) to determine the types of cells in the tissues and to detect the presence of starch. If starch is present in large amounts, it must be actively removed or else there may be more starch than cell walls, and the carbohydrate analyses will be meaningless. Some plants contain large amounts of starch in which almost every cell is packed full of granules (e.g., potato), while others contain small amounts of starch in which some of the cells contain granules (e.g., apples and unripe tomatoes), and still other plant tissues may contain very little or no starch (e.g., onions and pineapple). Even among the same plants, all material must be checked because there will be differences among varieties, stages of ripeness, and parts of the plants used.

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Problems encountered during the extraction, purification, and chromatography of pectic fragments, and some solutions to them

Cited by 121 publications

References 19 publications

Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening

Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening

Extraction of pectic polysaccharides from sugar-beet cell walls

Isolation of Plant Cell Walls and Fractionation of Cell Wall Polysaccharides

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