Surface tension analysis was carried out using the method of Makkar et al.
- Aqueous solutions of MC and PPGF were prepared at concentrations of 0.1, 0.5, 1, 2.5 and 5 mg/ml.
- Triton X-100 well known chemically synthesized surfactant was employed as a control.
- The surface tension of each sample was measured five times using a Du Nouy ring equipment and the average was calculated.
Assay for emulsification activity was carried out according to the modified method of Navon-Venezia et al.
- One hundred microliters of each hydrocarbons and olive oil, was added to 10 ml of 20 mM sodium phosphate buffer (from pH 5.0 to 7.5) or 20 mM Tris-HCl buffer (from pH 7.0 to 9.0) containing Surfactant, or Triton X-100 at 1 mg/ml in a 50 ml grass tube.
- Shaking reciprocally in a shaker at 30oC for 1 h and standing for 10 minutes.
- The lower phase was removed and absorbance was measured at 620 nm.
Assay for hemolytic activity was carried out according to the modified method Morán et al.
- Fresh human blood was mixed with an incomplete medium (1:1 v/v) containing (per liter): 10.4 g of RPMI 1640 Media, 5.95 g of HEPES, 0.5 g of L-glutamine, 2 g of NaHCO3, and 105 units of penicillin-G.
- The mixture was centrifuged at 3,000 rpm for 20 min at 4 Celcius degree and the supernatant was discarded.
- This procedure was repeated twice and the precipitate was resuspended in the medium (1:1 v/v).
- One hundred microliters of an aqueous solution of PPGF and protein fractions purified by electrophoresis was added into a mixture of 80 microliters of 20 mM sodium phosphate buffer containing 40 mM NaCl and 20 microliters of the red blood cell suspension described above containing approximately 18,000 cells in a 96-well microplate having a u-bottom.
- After incubation at 37ºC for 30 min, the minimum concentrations showing hemolytic activity were determined.
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