Comp. Biochem. PhysioL Vol. 102B, No. 2, pp. 325-329, 1992 0305-0491/92 $5.00 + 0.00 Printed in Great Britain © 1992 Pergamon Press Ltd INDIVIDUAL EXPRESSION PATTERNS OF ISOFORMS IN THE VENOM OF THE BOTHROPS ASPER MYOTOXIN SNAKE BRUNO LOMONTE and ELENACARMONA Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San Jost, Costa Rica (Received 14 October 1991) Abstraet--l. In order to investigate the distribution of myotoxin isoforms in the snake Bothrops asper, venoms from individual specimenswere analyzed by a cathodic electrophoretic system for basic proteins under native conditions. 2. The electrophoretic system resolvedat least fivebands with slight differencesin mobility, correspond- ing to the fastest migrating proteins in the venom. The identity of the bands was confirmed by immunoblotting, using a rabbit anti-myotoxin serum. 3. There were clear differences in the individual patterns of myotoxin isoform expression, both in specimensfrom the Atlantic and Pacificregions of Costa Riea. Some individualspossessed all fivevariants. 4. In agreement with previous reports, the venoms of ten newborn (less than I0 days of age) specimens completely lacked myotoxin bands, indicating an ontogenetic regulation in the expression of these toxins in B. asper. 5. One of the bands, corresponding to the lysine-49phospholipase myotoxin II, was the only isoform present in all individuals studied, suggestinga possible selective pressure for the conservation of this type of protein in the venom of B. asper. INTRODUCTION In many cases, detailed analyses of protein toxins isolated from snake venoms have revealed the exist- ence of a remarkable diversity of isoforms (Deems and Dennis, 1981; Rael et al., 1984; Nishida et al., 1985; Maraganore et al., 1984; Maraganore and Heinrikson, 1986; Bieber et aL, 1987; Faure and Bon, 1987; DuBourdieu et aL, 1987; Bieber and Johnson, 1988; Liu et aL, 1989; Griffin and Aird, 1990; Liu et aL, 1990). In the study of Bothrops asper venom, a basic phospholipase A2 with myotoxic activity was isolated and named B. asper myotoxin (Gutitrrez et al., 1984). Subsequent immunochemical studies using monoclonal antibodies against this toxin showed the existence of several cross-reactive isoforms with a similar molecular weight of 15,000-16,000, but with slight differences in charge (Lomonte and Kahan, 1988). Two of these isoforms have been isolated and their amino acid sequences determined. Myotoxin III (Kaiser et al., 1990) is biochemically similar to the original myotoxin, now referred to as myotoxin I, isolated by Gutitrrez et al. (1984). On the other hand, myotoxin II (Lomonte and Gutitrrez, 1989) is a lysine-49 phospholipase A2 variant with an extremely low enzymatic activity (0.1% of that of myotoxin III; Francis et al., 1991). All these myotoxin isoforms have been detected or isolated in studies utilizing venom pools from a large number of specimens of B. asper. Therefore, it was not known if all the variants are present in every individual, or if the diversity observed in venom pools reflects the sum of individual variations. The objec- tive of the present work was to address this question, analyzing venom samples obtained from single spec- imens by means of an electrophoretic system capable of resolving the different variants. MATERIALS AND METHODS Snake venoms Venom samples obtained from individual B. asper speci- mens were collected, centrifuged at low speed to remove cells and debris, and stored at -20°C. A total of 41 adult specimens from the Pacific and 17 from the Atlantic regions of Costa Riea were studid. The venom pools utilized for comparison were prepared at the serpentarium of the Insti- tuto Clodomiro Picado, from at least 30 specimens from each region, lyophilized and kept at -20°C. In one ease, it was possible to analyze venom obtained from a female snake and its offspring (ten newborns). Protein concentration of fiquid venom samples was determined by the Coomassie Blue method (Speetor, 1978), whereas lyophilized venoms were weighed on a microbalance (Cahn Instruments, CA). Electrophoresis The discontinuous cathodic eleetrophoresis system for basic proteins described by Reisfeld et al. (1962) was utilized, under native conditions. Slab polyacrylamide gels (12%) were run in a Bio-Rad protean cell (Bio-Rad Labora- tories, CA) at 100 V, until the tracking dye (basic fuchsin) reached the separating gel. Then, voltage was increased to 150V until the end of the run. The tank was cooled with circulating water at 12-15°C. Liquid venom samples were diluted I:10 with 0.06 M acetic acid, 0.06 M KOH, 28% glycerol, pH 6.8, and then 10/~1were applied on each lane. Proteins were stained with Coomassie Blue R-250. lmmunoblotting The myotoxin bands in Atlantic and Pacific-type venom pools were specificallydetected by immunoblotting (Towbin et aL, 1979), using a rabbit anti-B, asper myotoxin I serum (Lomonte et al., 1987). The venoms, separated by CaPB 102/2--1 325