Intra-specific Variation in the Protein Composition and Proteolytic Activity of Venom of Crotalus lepidus morulus from the Northeast of Mexico
The Tamaulipan rock rattlesnake (Crotalus lepidus morulus) is a small, cold-tolerant, mountain rattlesnake that occupies the Sierra Madre Oriental in southwestern Tamaulipas, central Nuevo Leon, and southeastern Coahuila in Mexico. The aim of the present study was to analyze and compare the protein profile and proteolytic activity of 16 individual and four pooled (representative of four regions Galeana, Santiago, Zaragoza, and Tamaulipas) venom samples of C. l. morulus from snakes collected in the northeast of Mexico. Individual and pooled venoms of C. l. morulus were analyzed by SDS-PAGE, gelatinolytic, and fibrinogenolytic assays. Additionally, the four pooled venoms were tested by 2-D electrophoresis and caseinolytic and hemorrhagic assays. Individually, venoms of C. l. morulus showed variation in their electrophoretic profile and proteolytic activity without an evident geographic trend. When comparing the pooled samples, venom from the south portion of the range (Zaragoza and Tamaulipas) showed higher proteolytic activity than samples from the central and north portion of the range (Galeana and Santiago, respectively). Furthermore, pooled venoms of C. l. morulus showed lower variation in electrophoretic profile than individual venoms. It is important to note that this is the first report of protein profile and enzymatic activities of venom of C. l. morulus.
Geographic origin of the venoms. The figure shows the altitude (A and B) and type of vegetation (B) of the different collect zones. (1) Monterrey, Nuevo Leon; (2) Santiago, Nuevo Leon; (3) Galeana, Nuevo Leon; (4) Zaragoza, Nuevo Leon; (5) Miquihuana, Tamaulipas; (6) Bustamante, Tamaulipas; (7) Jaumave, Tamaulipas.
Electrophoretic profiles of individual (A and C) and pool (B and D) of venoms of C. l. morulus in SDS-PAGE (9% SDS-polyacrylamide gel), under reducing (A and B) and non-reducing (C and D) conditions. (G) Galeana pool; (S) Santiago pool; (Z) Zaragoza pool; (T) Tamaulipas pool. Each lane was loaded with 25 µg of venom. Proteins were stained with Coomassie blue. Invitrogen (A and C) and Bio-Rad (B and D) molecular weight markers (kDa) are shown on the left and right of each image.
2-D electrophoresis of pooled venoms of C. l. morulus. Venoms (400 µg) were loaded in each gel. Proteins were stained with Coomassie blue. Molecular weight markers (kDa) are shown on the left and right of each image.
Zymography for evaluating the gelatinolytic activity of individual (A) and pool (B) of venom of C. l. morulus. (G) Galeana pool; (S) Santiago pool; (Z) Zaragoza pool; (T) Tamaulipas pool. Each lane was loaded with 10 µg of venom. The gels were stained with Coomassie blue. Invitrogen (A) and Bio-Rad (B) molecular weight markers (kDa) are shown on the left of each image.
Fibrinogenolytic activity of individual (A) and pooled (B1, B2), and (C) venom of C. l. morulus using human fibrinogen as substrate. (A) Individual venoms of C. l. morulus were incubated with human fibrinogen at 37°C for 60 min. (B1) Fibrinogenolytic activity of pooled venom samples of C. l. morulus at 37°C for 60 min without 10 mM EDTA and (B2) with 10 mM EDTA. (C) Fibrinogenolytic activity of pooled venom samples of C. l. morulus at different incubation times: 5, 15, 25, 35 min. (G) Galeana pool; (S) Santiago pool; (Z) Zaragoza pool; (T) Tamaulipas pool. Samples of fibrinogen/venom (25 µg) were electrophoresed under reduced conditions on a 12.5% gel and stained with Coomassie blue. Fib: human fibrinogen control. kDa: Molecular weight markers.
SDS-PAGE analysis of casein after digestion by pooled venoms of C. l. morulus. Venom samples were incubated with the casein solution at 37°C for 1 h in a ratio of 100∶10 (casein∶venom) with and without 20 mM EDTA. Lanes: 1) Casein; 2) Galeana pool; 3) Santiago pool; 4) Zaragoza pool; 5) Tamaulipas pool; 6) Galeana pool+EDTA; 7) Santiago pool+EDTA; 8) Zaragoza pool+EDTA; 9) Tamaulipas pool+EDTA.
Caseinolytic activity quantitative of pooled venoms of C. l. morulus. One unit of activity was defined as the amount of venom yielding an increase in O.D. of 1.0 per min at 280 nm. Specific activity was expressed as units/mg protein.
Hemorrhagic activity of venoms of C. l. morulus. Groups of three mice were injected (intra-dermal) separately with 25 µg of venom. After 3 h, mice were sacrificed by anesthesia and the dorsal surface of the skin was removed. (1) Control, (2) Control, (3) Galeana pool, (4) Santiago pool, (5) Zaragoza pool, and (6) Tamaulipas pool.
Contributor Notes
Associate Editor: D. S. Siegel.