Figure 5

AE partial separation by Sephadex G-50 M chromatography, mass spectrometry, fraction dose-respond, and effect on the action potential. (a) Fractions obtained from AE gel filtration chromatography. Sephadex G-50 M column (90 × 8 cm) was equilibrated with PBS. Absorbance was monitored at 280 nm and fractions were collected at a flow rate of 300 μl min⁻¹. Three absorbance peaks were detected and the corresponding fractions (FI, FII, and FIII) were collected and tested for effect on neuronal I_Nav monitored at − 20 mV (6–14 neurons in each case, held at − 100 mV and depolarized every 750 s for 20 ms). (b) The graph shows I_Nav inhibition time-course produced by FII (1 mg ml⁻¹; 11 neurons), while FI did not produce a significant effect (4 neurons; mean ± S.D.), the column graph resumes the inhibition produced by each fraction (1 mg ml⁻¹). (c) MALDI-TOF–MS spectrum obtained from the FII and FIII fractions obtained from AE gel filtration chromatography, the numbers indicated the mass for the main signals detected. In (d) FII was applied in distinct concentrations and a dose–response curve (in pink) was adjusted to data points indicating an EC₅₀ of 80 ± 16 μg ml⁻¹. (e) FII was tested on the neuronal action potential generated by depolarizing steps (35 ms, 0.75 nA) every 500 ms; three decreasing FII concentrations are illustrated. Traces in black (10 superimposed traces in each case) corresponded to control action potentials before FII superfusion, whereas red traces were obtained during FII application. 0.5 mg ml⁻¹ potently inhibited the action potential, a concentration 10 times lower still blocked completely the action potential in few seconds, while 0.01 mg ml⁻¹ FII was ineffective.