L 4200

The purified CGL1 was reduced with tri-n-butylphosphine in 7M guanidine-HCl, 10 mM EDTA, 0.5 M Tris-HCl pH 8.5. The reduced CGL1 was pyridylethylated with 4-vinyl pyridine at 25 °C for 4 h in the dark. The reduced and pyridylethylated CGL1 was chemically cleaved at the methionyl bonds with 1% CNBr in 70% (v/v) formic acid at 25 °C for 24 h by the method of Gross28 (link)35 (link)36 (link). The peptides generated by the CNBr cleavage were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) using HITACHI model L-6200 and L-4200 liquid chromatographs on a Wakosil-II 5C18 AR column (4.6 × 100 mm, Wako Pure Chemical Industries Ltd., Osaka, Japan). The column was equilibrated with solvent A (0.1% trifluoroacetic acid; TFA), and the peptides were eluted at the flow rate of 1 mL/min using a linear gradient of 0–100% solvent B (acetonitrile/water/TFA, 80:20:0.1 [v/v/v]) at room temperature. The separated fractions were collected for sequencing. Automated Edman degradation37 (link)38 39 (link)40 (link) was performed using a gas phase protein sequencer (Shimadzu model PPSQ-21).

The purified AJLec was reduced with tri-n-butylphosphine in 7 M guanidine-HCl, 10 mM EDTA, and 0.5 M Tris-HCl (pH 8.5) and pyridylethylated by treatment with 4-vinyl pyridine at 25 °C for 4 h in the dark. The resulting pyridylethylated AJLec was chemically cleaved at methionyl bonds with 1% CNBr in 70% (v/v) formic acid at 25 °C for 24 h by the method described by Gross^{31 (link)}. The peptides generated by CNBr cleavage were separated by reverse-phase HPLC using HITACHI model L-6200 and L-4200 liquid chromatographs on a Wakosil-II 5C18 AR column (4.6 × 100 mm, Wako Pure Chemical Industries Ltd., Osaka, Japan). The column was equilibrated with solvent A (0.1% trifluoroacetic acid; TFA), and the peptides were eluted at a flow rate of 1 mL/min using a linear gradient of 0–100% solvent B (acetonitrile/water/TFA, 80:20:0.1 [v/v/v]) at room temperature. Amino acid sequences of the separated peptides were determined using a PPSQ-21 protein sequencer (Shimadzu).