plant surface microbiology.pdf

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Donna M. Penrose and Bernard R. Glick
6.4 Measurement of ACC by HPLC
6.4.1 Chemicals
The Waters AccQ •Fluor Reagent Kit, AccQ•Tag eluent A concentrate (a premixed
concentrated acetate-phosphate buffer) and the amino acid standard, a
mixture of 17 amino acids (tryptophan, glutamine, and asparagine not
included) each at a concentration of 2.5 mM with the exception of cysteine
which is 1.25 mM, are supplied by Waters Limited. The Waters AccQ•Fluor
Reagent Kit contains the chemicals for derivatization: AccQ•Fluor reagent
powder (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; AQC), AccQ•
Fluor reagent borate buffer and AccQ•Fluor reagent diluent (acetonitrile).
ACC, b- and g-aminobutyric acid are purchased from Calbiochem-
Novabiochem Corp. (La Jolla, CA, USA), HPLC grade acetonitrile from Caledon
Laboratories (Georgetown, ON, Canada), a-aminobutyric acid from
Fisher Scientific, and L-a-(2-amino-ethoxyvinyl) glycine hydrochloride
(AVG) from Sigma-Aldrich Co. All water used is purified by a Milli-Q Water
System (Millipore Co. Bedford, MA, USA), autoclaved and then filtered
through a 0.45-mm HA filter (Millipore Co. Bedford, MA, USA).
6.4.2 Treatment of Glassware
All glassware used in this procedure is washed and then flushed at least six
times with tap water, twice with deionized water and twice more with distilled
water. Just prior to use, the cleansed glassware is wrapped in aluminum foil,
heated overnight at 275 °C and cooled to room temperature. Solutions and
samples are stored in heat-treated bottles and vials (including septa and lids).
6.4.3 Preparation of Standard Solutions
Stock 2.5-mM solutions of ACC,a-aminobutyric acid,b-aminobutyric acid,gaminobutyric
acid, and a mixture of 17 amino acids are prepared in 25 ml of
0.1 N HCl in a 25-ml volumetric flask. These solutions are diluted with sterile
distilled water to yield a concentration of 0.1 mM. The 2.5-mM and 0.1-mM
stock solutions are divided into 0.5-ml aliquots, frozen at –20 °C, thawed once
when needed and then discarded.With the exception of ACC,the 0.1-mM solutions
are further diluted with sterile distilled water to generate concentrations
between 5 and 25 pmol/20 ml injection. Dilutions of the 0.1-mM solutions of
ACC yield between 1 and 25 pmol ACC/20 ml injection. Standard mixtures of
ACC,a-,b-,and g - aminobutyric acids are prepared in sterile distilled water to
yield 12.5 pmol/20 ml injection. The amino acid standard is diluted such that
each 20-ml injection included 25 pmol of each of the 17 amino acids with the
exception of the amount of cysteine which was 12.5 pmol.Aliquots of the standard
solutions are frozen at –20 °C, and when required, thawed once and used.