Thus, fibrinolytic SVMPs could be useful to enhance skin re-epithelialization during wound healing. In conclusion, we described the presence of SVMPs and SVSPs in and venom extracts. measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. and extracts were the most proteolytic venoms among the samples. Particularly, predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we exhibited that spp. are a valuable source of proteases that Colchicine can aid chronic wound-healing treatments.  and bromelain from ) and microbial origins (collagenase from or vibriolysin from genus are of particular interest for exploration due to their greater proteolytic activity in comparison to other venomous snakes . Some snake venoms are specialized to generate the disruption of fundamental homeostatic processes. This venom specialization confers to the toxins a high affinity to a particular target, which converts each toxin into a potential source for design and development of new drugs . For this purpose, rattlesnakes (sp.) is the most representative genus of venomous snakes with 51 species described , 42 of which are distributed in Mexico . sp. venom contains many toxins, several of which are snake venom metalloproteases (SVMP) and snake venom serine proteases (SVSP). Together, both protease families represent about the 45% of the toxin abundance in their venoms and Colchicine can be as high as 93% (and specimens from the Chihuahuan Desert and Mexican plateau were extracted for testing. Protease characterization was performed using different quantitative and qualitative enzymatic assays including the universal proteolytic assay and zymography and with specific substrates, such as gelatin, L-TAME, fibrinogen, and fibrin. Using these techniques, we found that the venoms used in this study, specifically and snake venom proteases for the application as suitable wound-healing therapeutics. 2. Results 2.1. Crotalus Spp. Venom Toxin Families Identification by SDS-PAGE Colchicine First, we performed 12% SDS-PAGE with 15 g of each venom sample in order Colchicine to explore the venom protein profile. Venom banding patterns for and showed protein molecular weights from 12 to 116 kDa (Physique 1). All venoms tested contained comparable protein diversity at least through visual inspection of the 12% SDS-PAGE. venom was separated into 14 bands with different molecular weights (116, 75, 64, 52, 40, 31, 28, 23, 19, 16, 13.8, 13, 12.5, and 12 kDa). Very similar band profiles were observed for (116, 74, 70, 65, 46, 33, 27, 22, 19, 17, 14, 13, 12.7 and 12 kDa) and (106, 63, 59, 46, 29, 27, 24, 17, 14, 12.7, 12, and 11 kDa) with 12 protein bands. Lastly, displayed 13 bands of comparable molecular weights (106, 74, 68, 58, 48, 33, 27, 24, 19, 17, 16, 13, and 12 kDa). Open in a separate window Physique 1 SDS-PAGE venom banding profile for (COv), (CMNv), (CSv), and (CAv). 15 g of each venom were separated on a 12% SDS-PAGE and stained with Coomassie colloidal stain. Blue ovals enclose the typical molecular weight intervals for the most abundant toxin families according to Mackessy . 5-NT: 5-nucleotidases; CRiSP: cysteine-rich secreted proteins; LAAO: L-amino acid oxidases; PLA2: phospholipase A2; SVMP: snake venom metalloproteases; SVSP: Pde2a snake venom serine proteases. Additionally, we identified typical toxin families described previously  in all the venoms we analyzed (Physique 1, blue Colchicine ovals). In venom, we found a P-I SVMP and PLA2/C-type Lectin bands predominance, whereas and venoms had a higher abundance of the P-III SVMP band. Finally, venom showed a greater expression of SVSP and 5-NT/LAAO bands in comparison to the other venom samples. 2.2. Protease Inhibitors In order to confirm the nature of the proteases expressed in each venom, we performed universal protease activity assays using casein as the substrate together with several protease inhibitors, namely, EDTA,.