Protein Protocols Protein Protocols

568 Kochhar et al.
Fig.1. Marfey’s reagent (I) and L,L-diastereomer derivative from L-amino acid and the
reagent (II).
2. Materials
2.1. Vapor-Phase Protein Hydrolysis
1. Pyrex glass vials (25–50 × 5 mm) from Corning.
2. Screw-capped glass vials.
3. 6 N HCl from Pierce.
2.2. Derivatization Reaction
1. Amino acid standard solution H from Pierce.
2. L-Amino acid standard kit LAA21 from Sigma.
3. Marfey’s reagent from Pierce. Caution: Marfey’s reagent is a derivative of 1-fluoro-
2,4-dinitrobenzene, a suspected carcinogen. Recommended precautions should be
followed in its handling (16).
4. HPLC/spectroscopic grade triethylamine, methanol, acetone, and dimethyl sulfoxide
(DMSO) from Fluka.
2.3. Chromatographic Analysis
1. Solvent delivery system: Any typical HPLC system available from a number of
manufacturers can be used for resolution of derivatized amino acids. We have recently
used HPLC systems from Bio-Rad (HRLC 800 equipped with an autoinjector AST 100),
Hewlett-Packard 1050 system and Waters system.
2. Column: A silica-based C 8 reverse-phase column, for example, LiChrospher 100 RP 8
(250 × 4.6 mm; 5 µm from Macherey-Nagel), Aquapore RP 300 (220 × 4.6 mm; 7 µm
from Perkin-Elmer), Nucleosil 100-C 8 (250 × 4.6 mm; 5 µm from Macherey-Nagel), or
Vydac C 8 (250 × 4.6 mm; 5 µm from The Sep/a/ra/tions group).
3. Detector: A UV/VIS HPLC detector equipped with a flow cell of lightpath 0.5–1 cm and
a total volume of 3–10 µL (e.g., Bio-Rad 1790 UV/VIS monitor, Hewlett-Packard Photodiode
Array 1050, Waters Photodiode Array 996). The derivatized amino acids are
detected at 340 nm.