American Society of Hematology

Fig. 1.

$Fig. 1. Purification and determination of homogeneity of trocarin. (A) Gel chromatography of T carinatus venom on a Superdex 75 column. Flow rate: 1.5 mL/min. Procoagulant activities of all fractions were tested and fractions indicated by a horizontal bar were pooled. (B) Subfractionation of pooled procoagulant fraction by anion exchange chromatography on a UNO Q-1 column. Bound proteins were eluted by a linear gradient of NaCl. Flow rate: 2 mL/min. Procoagulant activities of all fractions were tested and fractions indicated by a horizontal bar were pooled. (C) RP-HPLC of fraction 6 from UNO Q-1 column. Column: Nucleosil C18. Bound proteins were eluted by a linear gradient of buffer B (80% ACN in 0.1% TFA). Flow rate: 2 mL/min. The procoagulant protein peak is labeled as trocarin. (D) Capillary zone electropherogram of trocarin. Electrophoresis runs of the protein (1 mg/mL) were performed on a BioFocus 3000 HPCE system (Bio-Rad) using a coated capillary (24 cm × 25 μm) from positive to negative polarities at 12 kV. Buffer: 100 mmol/L phosphate buffer, pH 2.5. Temperature: 18°C. Duration: 20 minutes. Sample injected using pressure mode 10 psi/s. Trocarin migrates as a single peak at 4.32 min.$

Purification and determination of homogeneity of trocarin. (A) Gel chromatography of T carinatus venom on a Superdex 75 column. Flow rate: 1.5 mL/min. Procoagulant activities of all fractions were tested and fractions indicated by a horizontal bar were pooled. (B) Subfractionation of pooled procoagulant fraction by anion exchange chromatography on a UNO Q-1 column. Bound proteins were eluted by a linear gradient of NaCl. Flow rate: 2 mL/min. Procoagulant activities of all fractions were tested and fractions indicated by a horizontal bar were pooled. (C) RP-HPLC of fraction 6 from UNO Q-1 column. Column: Nucleosil C18. Bound proteins were eluted by a linear gradient of buffer B (80% ACN in 0.1% TFA). Flow rate: 2 mL/min. The procoagulant protein peak is labeled as trocarin. (D) Capillary zone electropherogram of trocarin. Electrophoresis runs of the protein (1 mg/mL) were performed on a BioFocus 3000 HPCE system (Bio-Rad) using a coated capillary (24 cm × 25 μm) from positive to negative polarities at 12 kV. Buffer: 100 mmol/L phosphate buffer, pH 2.5. Temperature: 18°C. Duration: 20 minutes. Sample injected using pressure mode 10 psi/s. Trocarin migrates as a single peak at 4.32 min.

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