Amino acids analysis of axis and cotyledons of Pisum sativum by HPLC - Materials and Methods

Voir un cours sur la chromatographie.

Voir l'article (2004) Planta 219, 286 - 297.

Voir un cours sur les acides aminés.

  • Standards amino acid were from Merck
  • PITC, trifluoroacetic acid and triethylamine (sequanal grade) were from Fluka
  • acetonitrile (HPLC grade) was from Pierce

Derivatization of individual amino acids with PITC

PITC reacts with primary and secondary amines, leading to PTC-amino acids detectable at 254 nm (see figure below).

This method is sensitive down to at least 10 pmol (Bergman et al.).

  • standard amino acids plus homoserine (30 to 100 Ámol) were first neutralized with 1 mL ethanol:water:TEA (2:2:1, v/v) and evaporated under vacuum
  • samples were then derivatized by addition of 500 ÁL ethanol:water:TEA:PITC (7:1:1:1, v/v) corresponding to 5 times PITC excess (mol/mol)
  • samples were incubated for 30 min at room temperature and evaporated under vacuum
  • PTC-amino acids were dissolved in ethanol:water (at various ratio depending on the hydrophobic nature of the PTC-amino acid) and stored at - 20°C

amino acid analysis Medicago truncatula biochimej

Reference : Bergman, T., Carlquist, M., and Jörnvall, H. (1986) in Advanced Methods in Protein Microsequence Analysis (Wittmann - Liebold, B., Salnikov, J., and Erdmann, V. A., Eds.) 45 - 55, Berlin/Heidelberg

Extraction of amino acids from cotyledons and axis and derivatization with PITC

Cotyledons and axis were separated from seeds taken at various times of germination and amino acids were extracted from 20 to 30 organs.

In the case of the dried seed (i.e., 0 hour of imbibition) the whole seed was treated.

  • amino acids were extracted in 4 mL of water:ethanol (1:1) during 1 hour at 4°C under agitation, followed by a centrifugation (15000 g for 10 min). The supernatant was removed and evaporated under vacuum
  • for both types of organ, 8 mmoles of total amino acids were neutralized and derivatized with PITC in the same conditions as described for standard amino acids
  • samples were stored at - 20°C and were dissolved in water:ethanol (1:1) before HPLC analysis

HPLC analysis

Reverse phase-HPLC was performed using a Waters system (Millipore) with :

  • two Waters (Millipore) pumps 510
  • a Waters UV - visible detector 484
  • a Rheodyneinjection valve 7725i

System control and data processing were performed using Millenium 32 chromatography manager software (Waters).

Derivatized amino acids were loaded on a reverse-phase C18 Nova-Pak column 4 Ám (3.9 x 300 mm) thermostated at 37°C.

Click to see the complete system.

amino acid analysis Medicago truncatula biochimej

Derivatized amino acids were separated using two mobile phases :

  • solvent A : aqueous sodium acetate 20 mM, trifluoroacetic acid and triethylamine 0.04%
  • solvent B : acetonitrile
  • flow rate : 1.0 mL/min

Click to see the separation of the 20 PTC - amino acids

amino acid analysis Medicago truncatula biochimej

time (min) % A % B increase of B
0 93 7 --------
10 91 9 non linear
13 78 22 linear
23 68 32 non linear
26 20 80 non linear
29 93 7 non linear

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