Model 433a

Peptides were assembled using the Fmoc/tBu solid-phase peptide synthesis approach (39 (link)) using either model 433A (Applied Biosystems, CA, USA) or model Liberty (CEM Corporation, NC, USA) automated peptide synthesizers followed by cleavage in the trifluoroacetic acid (TFA)/phenol/thioanisole/ethanedithiol/water (10:0.75: 0.5:0.25:0.5, w/w) mixture at 25°C for 90 min followed by precipitation with cold diethyl ether. The crude peptides were purified by preparative reversed-phase high-pressure liquid chromatography (RP-HPLC). The peptide purity (>98%) was confirmed by analytical RP-HPLC, and the masses were confirmed by mass spectrometry. Following lyophilization, the purified peptides were obtained in the form of their TFA salts. Stock solutions (5 mg/mL) were prepared in ultrapure DNAse/RNAse free water (Thermo-Fisher, Loughborough, UK) and stored in aliquots at −80°C. The sequences of the peptides used in this study are shown in Table 1.

Peptides were synthesized from Fmoc-protected L-amino acid derivatives according to the method described by Kojima et al.[31] (link) by using an automated peptide synthesizer (model 433A, Applied Biosystems, California, U. S. A., 0.1 mmol scale with preloaded resin). After deprotection according to the manufacturer's protocol, each peptide was purified using a reversed-phase HPLC (Capcell Pak C18 column, SG, 10 or 15 mm i.d. × 250 mm; Shiseido Co., Ltd., Japan) with a linear elution gradient from 0.1 % trifluoro acetic acid (TFA) to 50 % or 70 % CH₃CN containing 0.1 % TFA over 30 min. The flow rate was set at 3 or 6 mL/min. The primary peak fractions were collected and then lyophilized. The purity of the synthetic peptides and the progress of the enzymatic reaction were confirmed by an analytical reversed-phase HPLC (Capcell Pak C18 column, MGII, 4.6 mm i.d. × 150 mm; Shiseido Co., Ltd., Japan) at a flow rate of 1.0 mL/min with a linear elution gradient from 0.1 % TFA to 70 % CH₃CN containing 0.1 % TFA. The column eluate was monitored with a photodiode-array detector (SPD-M20A; Shimadzu, Japan). Each purified peptide was characterized by ESI-MS using a Qstar Elite Hybrid LC-MS/MS system.

Peptides were prepared and their proteolytic activity was measured as described in our previous study [1 (link),2 ]. The peptides were synthesized (model 433A, Applied Biosystems, Foster City, CA, USA.; 0.1 mmol scale with preloaded resin). After deprotection according to the manufacturer’s protocol, each peptide was purified using reverse-phase preparative high-performance liquid chromatography (RP-HPLC), (Capcell Pak C18 column, SG, 15 mm i.d. 250 mm; OSAKA SODA Co., Ltd., Osaka, Japan) and the molecular weights of the peptides were confirmed by analytical HPLC (CapcellPak C18 column, MGII, 4.6 mm i.d. 150 mm; OSAKA SODA Co., Ltd., Osaka, Japan). Each purified peptide was characterized by electrospray ionization mass spectrometry using (ESI-MS) a Qstar Elite Hybrid LC–MS/MS system [1 (link),2 ].

The 45-residue sequence YKRCHKKGGHCFPKTVICLPPSSDFGKMDCRWKWKCCKKGSVNNA-COOH was obtained by the solid-phase method using a peptide synthesizer (Model 433A, Applied Biosystems). The synthetic myotoxin-3 was incubated in a 0.1 M Tris (pH 8.3)-ACN (70-30, v/v) solution to allow the oxidation of the six cysteines and the formation of three disulfide bonds. The oxidized peptide was then purified by RP-HPLC, and the oxidation was confirmed by comparing the mass by MALDI-TOF of the non-oxidized synthetic peptide obtained by MALDI-TOF versus the oxidized synthetic peptide.