Amino
acid
and
amino
sugar
assay
The fragmented thalli were washed in 0,5 M sodium phosphate
butTer
(pH 7,5) and
lyophilized. Ten
mg
of
thallus material was hydrolyzed in 1 cm
3
6N HCI at 100°C
for
16
h
in
a sealed ampule. The solution was then evaporated to dryness in a rotary
flask evaporator and finally redissolved in 1 cm
3
distilled water. The pH was adjusted
to pH 8,0 with 5N NaOH. The solution was centrifuged and the clear supernatant
stored at 4°C.
the
solutions
(0,1
cm
3
)
were diluted with 0,9
cm
3
citrate
butTer
(pH 2,2) and 0,2 cm
3
of
this was used for assay on a Bio-Cal BC-200 automatic,
amino acid analyser. Resins used in the assay were
of
the Aminex polystyral base type
with a cross-linking
of
7,5 to 8,0% divinylbenzene. The assay specifications were as.
follows:
1.
Basic amino acids
Column
Resin
Resin height
ButTers
Temperature
25
cm with internal diameter
of
0,9 cm
Arninex AS, particle size 11,5-15,5
~m
14
cm
- pH 5,28; 0,35N
in
Na
ions and 0,2N
NaOH
- 50°C
2.
Neutral and acidic amino acids
Column 60 cm with internal diameter
of
0,9 cm
Resin - Aminex A6, particle size 15,5-19,5
~m
Resin height - 54 cm
ButTers
- A = pH 3,25; 0,2N in
Na
ions
B
= pH 5,25; 0,2N
in
Na
ions and 0,2N
NaOH
Temperature - 50°C
An amino acid calibration mixture supplied by Beckman Instruments was used as
standard. This standard solution contained 1 cm
3
of
0,62N HCI with 2,50 ± 0,004
~mole
of
each amino acid. One cm
3
of
this solution was diluted to
five
cm
3
and
0,2 cm
3
was applied to the
colu~n,
giving a final concentration
of
0,1
~mole.
Due to
the low solubility
of
cystine it was only present in half the concentration. The amount
of
each amino acid was determined by the width x height method
as
described in the
manual. During prolonged hydrolysis with 6N HCI, the amino sugars underwent
hydrolytic degradation.
No
attempt was made to correct for the hydrolytic loss
of
the
amino sugars or the corresponding increases in ammonia.
Analysis
of
the
cell
sugars
A modification
of
the method
of
Cato, Cummins and Smith (1970) was used for
tl~e'
analysis
of
the
cell
sugars. Ten
mg
amounts
of
washed
thal,li
were hydrolyzed in 2
C~3'
of
2N H2S04 for two hours in a boiling water bath. When cooled, the acid
walneu-
tralized with solid barium hydroxide to a pH
of
7,0.
The~recipitate
was removed by
centrifugation and the deposit washed with 2 cm
3
of
distilled water. The supernatant
was evaporated in a water bath at 60°C. The dry
residu~
was dissolved
in
0,3 cm
3
distilled water. The solution at this step was clear
of
any remaining barium hydroxide.
One dimensional ascending thin layer chromatography
wa~
performed on silica
gel
G
38
Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2012)
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