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

摘要

The venom of Crotalus molossus molossus (blacktailed rattlesnake) is very basic compared to that of other Crotalinae venoms. Unlike other Crotalinae venoms that are separated by anion exchange chromatography, C. m. molossus venom must be fractionated by cation exchange chromatography. Electrophoretic titration (ET) was used to predict the isoelectric point (pI) and optimal conditions for isolation. The specific hemorrhagic activity for C. m. molossus venom was 7.5 mm/mug, making it one of the most hemorrhagic of Crotalinae venoms. Basic hemorrhagic and fibrinolytic proteins from the venom of C. m. molossus venom were further fractionated by cation exchange chromatography. A basic fibrinolytic/hemorrhagic protein (CMM4) was isolated. CMM4 has 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 very similar surface charges at all pH. CMM4 did not activate plasminogen when tested with a Chrom Z-PLG assay. The proteins in CMM4 had similar N-terminal 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 are in positions 8 and 10. CMM4 may contain isoforms that differ by minor sequence variations at their amino-termini. The amino acid sequences of CMM4 were very similar to other fibrinolytic and hemorrhagic metalloproteinases isolated from venoms of the genera Crotalus. The specific hemorrhagic activity of CMM4 decreased as the specific fibrinolytic activity increased. A monoclonal antibody (CMM1b) was produced against C. m. molossus venom that neutralized the hemorrhagic activity of some of its fractions. CMM1b also reacted with 11 of 29 venom samples tested via ELISA. (C) 2000 Elsevier Science Ltd. All rights reserved. [References: 53]