Proteolytic Assays

Fibrinolytic

  • Fibrinolytic activity of venom fractions is measured using a procedure modified from Bajwa et al. (1980).

  • Fibrinogen solution (9.4 mg/ml, 300 µl) and thrombin solution (38.5 U/ml, 10 µl) are added to each well of a 24-well plate.

  • The plate is shaken gently and the solution allowed to clot at room temperature.

  • The plate is then incubated for 3 hr at 37˚C. Twenty microliters of each venom fraction are added individually in the center of their corresponding wells and incubated overnight at 37˚C.

  • Seven hundred microliters of 10% trichloroacetic acid (TCA) are placed in each well, and decanted after 10 min.

  • If a zone of clearing is observed the assay is considered positive.

  • The assay is repeated three times for each fraction.

Fibrinogenolytic

  • A modified method of Ouyang and Teng (1976) is used to test the fibrinogenolytic activity of venom fractions.

  • A 20 microliter solution of human fibrinogen (10 mg/ml) in 50 mM Tris/HCl buffer, pH 8.5, and 10 microliters of the sample (0.03 mg/mL) are mixed and incubated at 37˚C for 2 h.

  • Twenty microliters aliquots are taken and added to 20 µl of 20 mM Tris/HCl buffer, pH 8.0, containing 2 mM EDTA, 5% (w/v) SDS, 0.02 % (w/v) bromophenol blue and 10% (v/v) beta-mercaptoethanol.

  • A volume of 0.3 µl of sample is applied to a Homogenous 12.5 PhastGel and separated by a Pharmacia PhastSystem.

  • The gels are stained using silver nitrate.

Hide Powder Assay

  • A modified method of Rinderknecht et al. (1968) is used to test for proteolytic activity in the venom.

  • Hide powder azure is ground with a mortar and pestle to obtain uniform consistency.

  • Eight milligrams of this powder are then mixed with 2 ml of 0.02 M Tris-HCl buffer, pH 8.0, and 100 µl of each venom fraction are added to the vial.

  • Each vial is incubated for 1 hr at 37˚C, and agitated at 5 min intervals.

  • After incubation, each vial is centrifuged at 420 x g for 5 min.

  • The supernatant is transferred into a different vial and the absorbance measured at 595 nm.

  • The hide powder azure assay is considered positive if an absorbance reading greater than 0.1 is obtained.

  • The assay is repeated three times..

Gelatinase Assay

  • Gelatinase activity of the venom fractions is tested using a method modified from Huang and Pérez (1980).

  • Twenty microliters of each venom fraction are placed on a Kodak X-OMAT™ scientific imaging film having a gelatin coating.

  • Clearing of gelatin on the X-ray film is determined by washing the film with tap water following incubation at 37˚C for 4 hr in a moist incubator.

  • A resulting clear spot on the X-ray film indicated that venom HPLC fraction is positive.

  • The assay is repeated three times.