I. Isolation of Cell Walls

II. Cellulose Determination; Total Sugar and Uronic Acids

III. Fractionation of Cell Wall Polysaccharides

IV. Methyl Esterification of Uronic Acids

V. Monosaccharide Composition

VI. Linkage (methylation) Analysis

VII. Fourier Transform Infrared (FTIR)

VIII. Protocol for the screening of the UniformMu maize population with near infrared reflectance spectroscopy


IV. Methyl Esterification of Uronic Acids

A. Determination of uronosyl methyl esters, methanol content after saponification

Principle: Uronosylmethyl esters are saponified to yield methanol and the free acids- With a modified procedure of Wood and Siddiqui [Analyt. Biochem. 39 (1971) 418-428] methanol is oxidized to formaldehyde and detected subsequently with a pentane-2,4-dione to form a yellow chromagen. Paired assays of uronic acid give the proportion of methylesterified uronosyl esters.

Reagents:


1.5 M NaOH (6 g/100 mL)

4.5 M H2S04 (25.3 mL analytical reagent added to 50 mL of water; cool, bring to 100 mL)

2% KMn04 (2 g/1OOmL of H2O, filter through fine sintered glass funnel and store in dark bottle)

0.5 M Na arsenite in 0.06 M H2S04.

0.02 M pentane-2,4-dione in 2.0 M NH40Ac/0.5 M HOAc (make fresh every two weeks)
i) Add 0.2 moles NH4OH (13.5 mL of 14.8 M stock) to 50 mL of water gently stirring in a
100-mL beaker in an ice-bath. Then add 0.25 moles gl. acetic acid (14.4 mL of 17.4 M
stock). After the solution cools to ambient bring to 100 mL. ii) Tare a 100-mL graduated
cylinder on the Mettler 360. Pipet 0.202 g ofpentane-2,4-dione into cylinder then bring to
100 mL with the fresh 2.0 M NH4OAc/0.5 M HOAc.

Saponification:

1. Weigh solid material and add to a 1.5-mL Eppendorf centrifuge tube and add 0.75 mL of H2O (or add 0.75 mL of an aqueous suspension. [For pectins, i. e. an ammonium oxalate extract use 1 to 2 mg of lyophilized material; for total cell wall. use 5 to 10 mg]. Standard curve can be made with PGA methyl ester standards or dilutions of 1 M MeOH (see below).

2. Add 0.25 mL of 1.5 M NaOH, vortex, and let stand at ambient temperature for 30 min with occasional vortexing.

3. Chill Eppendorfs on ice and then add 0.25 mL of 4.5 M H2S04. Vortex quickly and return to ice for 10 min. Micro fuge for 5 min in cold room.

4. Carefully remove by pipet 1.0 mL of the clear solution above the pellet and add to a 6-mL tube on ice. Don't dribble the solution down the sides but pipet directly to the bottom of the tube.

Assay:

1. To the 1.0 mL of acidified saponification supernatant add 200 µL of 2% KMNO4 directly to solution. Do not run the KMN04 down the sides of the tube and don't vortex. Keep chilled on ice for 15 min.

2. Add 200 µL of Na arsenite reagent and now vortex quickly and let stand at ambient
temperature for at least 60 min.

3 . Add 2 mL of the pentane-2,4-dione reagent and vortex quickly; Heat tubes capped with marbles at 60°C for 15 min for full color development. Read the yellow chromagen at 412 nm.

Standards:

For methanol standard curve: Make 1 M MeOH from analytical reagent and store in glass-tight bottle. Make dilution series to range from 50 to 2000 nmol MeOH/mL and carry through saponification like the PGAs and wall materials.

B. Determination of Uronic Acids (see Section II)

Use ratio of nmole methanol / nmole uronic acid to determine dgree of etherification.

 

 



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