Manufacturing Process
Triethylaminoethyl cellulose powder (Serva) of capacity 0.71 m.equiv./gm is
suspended in 2 M sodium chloride buffered with 0.1 M tris/phosphate pH 6.0
and the slurry is packed into a glass column 3.6 cm in diameter until the
height of the packed material reaches 20 cm. The column is washed with a
further 2 L of the solvent used for preparing the slurry and is then
equilibrated with 0.01 M tris/phosphate buffer pH 8.5. Tris is an abbreviation
for tris(hydroxymethyl)aminomethane. 330-360 mg of crude A. rhodostoma
venom is dissolved in 20 ml of 0.01 M tris/phosphate buffer pH 8.5,
centrifuged to remove insoluble material, and the clear supernatant is applied
to the column. The fractionation is carried out at room temperature at a flow
rate of 90-100 ml (35 ml/hour). The protein concentration in the eluate is
estimated from the extinction of the solution at 280 m/t in 1 cm cells. The
chromatogram is developed with the following buffers. In all cases the
molarity of the buffers are with respect 40 to tris.
0.01 M tris/phosphate pH 8.5 (to wash venom onto the column) (fractions 1,
2, 3). 0.01 M tris/phosphate pH 7.0 (fraction 4), 0.02 M tris/phosphate pH
6.0, 0.04 M tris/phosphate pH 6.0 (fraction 5), 0.10 M tris/phosphate pH 6.0
(fraction 6), 0.10 M tris/phosphate + 0.10 M NaCl pH 6.0 (fraction 7).
The changes in eluting buffer are made after the column has equilibrated with
the buffer. The protein fractions obtained in this way are assayed for
coagulant activity.
Less than 1% of the applied coagulant activity is recovered in fraction Nos. 1,
2, 3, 4. Fraction 1 however possesses proteolytic activtity which in
concentrated solutions would dissolve fibrin clots. The thrombin-like activity is
eluted from these columns in significant amounts at a buffer strength of 0.04
M or greater.
The eluate is freeze dried to give a light powdery product. Yield: 18-20 mg
per 350 mg dry venom. Calculation of molecular weight of the protein based
on the amino acid composition gives a value of 30.000 which is in a very good
agreement with that obtained by physical methods.
