5. Samples of this material
(ca 4.5 mg) were further subjected to hydrophobic interaction HPLC on a HiTrap Butyl HP column (1.6 × 2.5 cm, from GE Healthcare, Uppsala, Sweden) equilibrated with 100 mM PB containing 1 M (NH4)2SO4, pH 7.5. After sample application, the column was eluted with a segmented gradient of 1.0–0 M (NH4)2SO4 in the same buffer at 1 mL/min flow rate. The fractions were collected manually; the selected cytolytic fractions were combined. The buffer of the active samples was exchanged to PBS using an Amicon® Ultra device (cut-off 10 kDa) at 4 °C. As for the last step, this material (ca 700 μg) had its NaCl concentration adjusted to 0.1 M and was loaded on a Synchropak ABT 199 http://www.selleckchem.com/products/MDV3100.html SAX 300 (Eprogen, USA) anion exchange HPLC column (250 × 4.6 mm), previously equilibrated with 20 mM PB, 0.1 M NaCl pH 7.5 and eluted with a segmented gradient of the equilibrium buffer added by 1 M NaCl at 0.5 mL/min flow rate. The fractions were collected manually and the purified hemolytic fraction, referred to as Sp-CTx, was concentrated using Amicon Ultra device (as mentioned above), stabilized with glycerol (10%
v/v) and stored at −196 °C until required. The degree of purity of the hemolytic samples was assessed by SDS-PAGE according to Laemmli (1970). Hemolytic activity was assayed on rabbit erythrocytes, which are highly sensitive to fish venoms (Kreger, 1991 and Shiomi et al., 1989). Rabbit blood was collected
by cardiac puncture and mixed with Alsever’s solution (1:1 ratio). To detect the hemolytic activity during the purification procedure, samples of crude venom and purified fractions were incubated with washed erythrocytes suspension (2% v/v) in phosphate buffered saline (PBS) for 10 min at 25 °C and were centrifuged (14,000 g for 1 min) at room temperature. The amount of hemoglobin released in the supernatant was measured spectrophotometrically at a wave length of 540 nm. Total hemolysis was determined by incubating the erythrocytes suspension in distilled water. An osmotic protection assay was carried out to investigate if the formation of pores by Sp-CTx in the cell membrane is involved in the hemolytic effect of this toxin. Washed rabbit erythrocytes Carnitine palmitoyltransferase II were obtained as described above. For this experiment, saccharose and polyethylene glycol (PEG) of different molecular sizes (1000, 1450, 3350 and 8000 with SEr – Stokes–Einstein hydrodynamic radius of 1.0; 1.2, 1.9 and 3.2 nm, respectively) (Kuga, 1981) was added to hemolytic assay buffer at the final concentration of 30 mM and the percentages of hemolysis inhibition were calculated. The incubation period of rabbit erythrocytes with Sp-CTx (50 ng/mL, 2× EC50) was up to 120 min. The time course of erythrocyte lysis induced by Sp-CTx was followed spectrophotometrically at 700 nm at room temperature. The initial A700 was approximately 0.9.