EVIDENCE FOR ISOMERIZATION IN MYOTOXIN-A FROM THE PRAIRIE RATTLESNAKE(CROTALUS-VIRIDIS-VIRIDIS)

Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridi s viridis, exists asa temperature-dependent equilibrium of two interco nverting forms. Reverse-phase high-performance liquid chromatography ( RP-HPLC) shows that the two forms interconvert slowly enough at 25 deg rees C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same t wo peaks in the same ratio of areas. The two peaks merge during chroma tography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5 degrees C, the individual peaks can be isolated and maintained for several days without reachin g equilibrium. Mass analysis by matrix-assisted laser desorption ioniz ation (MALDI) time-of-flight mass spectrometry shows that myotoxin a i s present in both RP-HPLC peaks, suggesting that the two resolved form s are conformational isomers. Capillary zone electrophoresis (CZE) als o shows two resolved, but interconvertible peaks over a range of pH va lues. Furthermore, RP-HPLC chromatograms of myotoxin a at concentratio ns from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional H-1-NMR nuclear Ove rhauser enhancement spectroscopy indicates the presence of a cis-proli ne peptide bond, consistent with an equilibrium mixture of cis-trans i somers; however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isola ted at c. 5 degrees C. (C) 1996 Elsevier Science Ltd.