Partial characterization of a basic protein from Crotalus molossus molossus (northern blacktail rattlesnake) venom and production of a monoclonal antibody

The venom of Crotalus molossus molossus (blacktailed rattlesnake) is very b asic compared to that of other Crotalinae venoms. Unlike other Crotalinae v enoms that are separated by anion exchange chromatography, C. m. molossus v enom must be fractionated by cation exchange chromatography. Electrophoreti c titration (ET) was used to predict the isoelectric point (pI) and optimal conditions for isolation. The specific hemorrhagic activity for C. m. molo ssus venom was 7.5 mm/mug, making it one of the most hemorrhagic of Crotali nae venoms. Basic hemorrhagic and fibrinolytic proteins from the venom of C . m. molossus venom were further fractionated by cation exchange chromatogr aphy. A basic fibrinolytic/hemorrhagic protein (CMM4) was isolated. CMM4 ha s a molecular weight between 23 and 26 kDa and a pI of approximately 11.3. SDS electrophoresis revealed one band and ET curve revealed 3 bands with ve ry similar surface charges at all pH. CMM4 did not activate plasminogen whe n tested with a Chrom Z-PLG assay. The proteins in CMM4 had similar N-termi nal amino acid sequences to each other (D-Q-Q-N-L-P-Q-(S/A/R)-Y-(V/R/I)-E-L -V-V-V-A-D-H-R-L-F-M-K-Y-K-S-D-L-N-T). The differences in these proteins ar e in positions 8 and 10. CMM4 may contain isoforms that differ by minor seq uence variations at their amino-termini. The amino acid sequences of CMM4 w ere very similar to other fibrinolytic and hemorrhagic metalloproteinases i solated from venoms of the genera Crotalus. The specific hemorrhagic activi ty of CMM4 decreased as the specific fibrinolytic activity increased. A mon oclonal antibody (CMM1b) was produced against C. m. molossus venom that neu tralized the hemorrhagic activity of some of its fractions. CMM1b also reac ted with 11 of 29 venom samples tested via ELISA. (C) 2000 Elsevier Science Ltd. All rights reserved.