Mojave toxin: rapid purification, heterogeneity and resistance to denaturation by urea.
This report establishes that purified Mojave toxin prepared from the snake venom of Crotalus scutulatus scutulatus contains multiple heterogeneous dimers (isoforms) differing slightly in isoelectric points. This conclusion is based upon chromatographic, immunological, sodium dodecyl sulfate--polyacrylamide gel electrophoretic and polyacrylamide isoelectric focusing experiments. The Mojave toxin-related proteins were rapidly purified from venom via a single chromatography step. Generation of Mojave toxin-related proteins from isolated subunits and immunoblots of these proteins subsequent to electrophoretic separation demonstrate that each of the proteins consists of acidic and phospholipase basic subunits. The analysis of venom in narrow range polyacrylamide isoelectric focusing gels at varying concentrations of urea, in conjunction with immunoblots utilizing antibodies specific to the basic subunit, demonstrates that the isoforms of Mojave toxin are native and not artifacts from isolation procedures. Analyses of venoms from Crotalus scutulatus scutulatus individuals indicate that each snake produces multiple isoforms of the neurotoxin. Additionally, the same predominant isoform of Mojave toxin is present in both individual and commercial venoms. The heterogeneity of the Mojave toxin-related proteins is largely due to differences in the acidic subunits and some of the forms may reflect post-translational processing of the protein. The Mojave toxin-related proteins demonstrate a resistance to urea denaturation by characteristically entering and focusing in polyacrylamide isoelectric focusing gels containing 0-6 M urea, but dissociating to constituent subunits in 8 M urea. Experimental evidence suggest that salt bridges may be important in stabilization of the Mojave toxin complex.
Johnson
G R
GR
Department of Chemistry, Arizona State University, Tempe 85287-1604.
Bieber
A L
AL
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000032:analysis / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D009498:Neurotoxins / Q000032:analysis / Q000302:isolation & purification*; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate; D014508:Urea
D003435:Crotalid Venoms; C018107:Mojave toxin; D009498:Neurotoxins; D012967:Sodium Dodecyl Sulfate; D014508:Urea