Protein Protocols Protein Protocols

570 Kochhar et al.
Table 1
HPLC Program for Resolution of Amino Acids Derivatized with Marfey’s Reagent
Time (min) % Solvent A % Solvent B Input
0 90 10
15 90 10 Detector auto zero
15.1 90 10 Inject
115 50 50
165 0 100
170 0 100
Fig. 2. Separation of 2,4-dinitrophenyl-5-L-alanine amide derivatives of L-amino acids by
HPLC. A 20-µL aliquot from the amino acid standard mixture (standard H from Pierce Chemical
Company) was derivatized with Marfey’s reagent. Chromatographic conditions: Column,
LiChrospher 100 RP 8 (250 × 4.6 mm; 5 µm from Macherey-Nagel); sample, 100 pmol of
diastereomeric derivatives of 18 L-amino acids; solvent A, 13 mM trifluoroacetic acid plus 4%
(v/v) tetrahydrofuran in water; solvent B, 50% (v/v) acetonitrile in solvent A; flow rate, 1 mL/min;
detection at 340 nm; elution with a linear gradient of solvent B in solvent A (Table 1). The
amino acid derivatives are denoted by single-letter code, cysteic acid as CYA and cystine as
Cs. FFDA indicates the reagent peak; the peaks without denotations are reagent-related as
shown in an independent chromatographic run of the reagent alone.
4. Notes
1. For analysis of amino acids as substrates or products in an enzymic reaction, deproteinization
is required. Add 4 M perchloric acid to a final concentration of 1 M and incubate
on ice for at least 15 min. Excess perchloric acid is precipitated as KClO 4 by adding an
equal volume of ice-cold 2 M KOH. After centrifugation for 15 min at 4°C, the supernatant
is collected, dried, and used for derivatization.
2. Complete removal of HCl is absolutely essential for quantitative reaction between amino
acids and Marfey’s reagent.