Sunday, March 29, 2009

Surfactant Properties: How to Quantitatively Measure

In order to measure the properties of surfactant, we can use the surface tension, emulsification activity, and hemolytic activity as the parameters. Here are the methods:

Surface tension analysis was carried out using the method of Makkar et al.
  1. Aqueous solutions of MC and PPGF were prepared at concentrations of 0.1, 0.5, 1, 2.5 and 5 mg/ml.
  2. Triton X-100 well known chemically synthesized surfactant was employed as a control.
  3. 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.
  1. 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.
  2. Shaking reciprocally in a shaker at 30oC for 1 h and standing for 10 minutes.
  3. 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.
  1. 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.
  2. The mixture was centrifuged at 3,000 rpm for 20 min at 4 Celcius degree and the supernatant was discarded.
  3. This procedure was repeated twice and the precipitate was resuspended in the medium (1:1 v/v).
  4. 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.
  5. After incubation at 37ºC for 30 min, the minimum concentrations showing hemolytic activity were determined.