Biology Reference
In-Depth Information
Separate completely acetylated PIA from other molecules on a Superdex 200
10/300 GL column, partially or completely deacetylated PIA on a Jordi PolarPac
WAX 10,000A 300 × 7.8 mm column (
see
Note 7
).
3.4.2. Colorimetric Detection of the Degree of PIA Deacetylation
by Analysis of Free Amino Groups
First, create a standard curve by using completely acetylated or deacetylated
PIA as calibration standard (0-1 mg/mL) (
see
Note 8
).
1. Add 100 μL of 4% NaHCO
3
(pH 8.5) and 100 μL of 0.1% TNBS to 100 μL of
sample.
2. Allow mixture to react at 40 °C for 2 h.
3. Add 50 μL HCl to stop the reaction and measure the absorbance at 335 nm with
a UV spectrophotometer.
4. Notes
1. Recent achievements in molecular biology have created multiple methods that
can be utilized to construct mutants by allelic replacements in different bacteria.
A variety of temperature-sensitive plasmids, antibiotics resistance markers, and
transformation protocols are now available.
2. Boiling cells with 0.5
M
EDTA, pH 8.0,
(5)
is the best method known to date for
the isolation of crude PIA from the bacterial cell surface and might be applicable
for staphylococcal cells only. PIA-related polysaccharide polymers have been
purified from
E. coli
by incubating cells in 50 m
M
Tris buffer, pH 8.0, 100 mg
lysozyme, and 0.1
M
EDTA at room temperature for 2 h
(7)
.
3. PIA is a -1,6 linked
N
-acetyl glucosamine homopolymer. Deacetylated PIA
is cationic because of the free amino groups with a theoretical pK = 6.9 that
become protonated at neutral or acidic pH. Acetylated PIA is insoluble at neutral
pH, especially after precipitation from the culture filtrate with ethanol, or with
increasing concentration of PIA after reducing the volume by ultrafiltration
devices.
4. The initial PIA purification method was developed by Mack et al.
(3)
. These
authors used a different, two-step chromatography protocol involving size-
exclusion and ion exchange chromatography on Sephadex G-200, Q-Sepharose,
and S-Sepharose. A similar purification method has been described recently to
isolate a PIA-related polysaccharide polymer in
E. coli
(7)
. Briefly,
E. coli
cells
were incubated in 50 m
M
Tris-HCL buffer (pH 8.0), 100 mg lysozyme, and
0.1
M
EDTA at room temperature for 2 h. Phenol/chloroform extraction steps
were performed to separate protein and debris contamination from the polysac-
charide. Samples were concentrated by ultrafiltration devices (10,000 MW cut
off) and fractionated on a fast protein liquid chromatography (FPLC) system with
a Sephacryl S-2000 column (equilibration and elution buffer: 0.1
M
PBS, pH 7.4).
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