OISHI, N.Y.; UEDA, C.M.P.M.; DIAS, W. de O.; LATRILHA, C.M.L.; RAW. I, The possible role of 
para-pepsin in the preparation of anti-venom sera. Mem. Inst. Butantan, 49 (21:41-45, 1987. 
ted with ammonium sulfate and heat desnaturated according to Harms 1 . 
Anti-venom assay: Antitoxic activity was assayed by mixing a range of 
venom concentrations with anti-venom in vitro, and administered intrave- 
nously to 150-200g chickens. The activity is expressed as the maximum, in 
mg of venom that is neutralized, having within 24 hours, no letal effect. 
Pepsin activity determination: 1 ml of a aqueous solution of pepsin, 
containing up to 100 mg of pepsin/L, was incubated with a 5 ml of 0.6% 
casein in 0.02N hydrochloric acid, that unless otherwise stated, adjusted to 
pH 2.3. Incubation was carried out for 30 minutes at 37° C, and the reac- 
tion stopped with 5 ml of 18g of trichloroacetic acid, 18g of sodium acetate 
and 20 ml of glacial acetic acid in 100 ml. After centrifugation, the superna- 
tant was read at 275 nm, using a tyrosine solution as standard. Results are 
expressed in enzyme units, one unit being defined as the amount of enzy- 
me that causes the release of 1 ^g of tyrosine in 30 minutes. 
Pepsin purification: Pepsin was purified as described by Ryle and 
Porter 7 : 500 g of crude pepsin was dissolved in 1 L of 0.5N sulfuric acid, and 
the insoluble materiais removed by centrifugation. The supernatant was 
precipitated by the addition of 1 L of saturated magnesium sulfate solution, 
and the precipitate collect by centrifugation and redissolved in water. 
Pepsin fractionation on DEAE-cellulose: Purified pepsin was chromato- 
graphed on DEAE-cellulose as described by Ryle and Porter 7 , or by a batch 
treatment by which the pepsin solution at pH 4.0 was treated with an equal 
volume of 100 g/L of DEAE-cellulose suspension equilibrated in 0.1 M ace¬ 
tate buffer pH 4.0. The non-absorbed fraction (A) was removed by filtra- 
tion, and the DEAE-cellulose sucessively eluted with 0.1 M and 0.2 M so¬ 
dium chloride in 0.1 M acetate buffer pH 4.0 (fractions B and C). 
Complement fixation: was carried out according to standart methods 
(Nowortny 2 ). 
Protein concentration was determined by a modification of the Lowry 
method (Petterson 3 ). 
RESULTS AND DISCUSSION 
Table 1 shows the effect of pepsin concentration on the yield of anti- 
crotalic serum. As already described by Pope 4 and Harms 1 , a good yield re- 
quires a large amount of pepsin, and a longer digestion time will not replace 
the high enzyme concentration. Table 2 shows that the concentration re- 
quired for the removal of Fc, with the inibition of complement fixation, can 
be achieved with about one thousand of the pepsin concentration. The 
transformation of the immunoglobulin in a F(ab) 2 i s not the limiting factor 
in the pepsin treatment, but the liberation of a hydrolyzis product that is 
heat stable, and thus resists the purification process (Pope 4 ; Pope and Ste- 
vens 6 ) 
lt is clear from te comparison of different commercial pepsin prepara- 
tions (table 3), that there is no relation between the caseinolytic activity 
measured at pH 2.3 and the anti-venom yield. It was suggested by Pope 
and Stevens 6 , that crude pepsin contained several other enzymes, and that 
the anti-venom hydrolysis was due to a cathepsin type enzyme present in 
crude pepsin. Ryle and Porter 7 found by DEAE-cellulose chromatography 
the existance of two pepsin-like enzymes, that were named para-pepsins, 
which differ from the main pepsin in their optimal pH. 
