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Abstract
Prior studies have indicated that highly purified samples of natural fibrolase. a fibrinolytic metalloproteinase from southern copperhead snake venom, contains two isoforms. The isoelectric points of these isoforms differ by 0.01 to 0.03 pH units. In this study we show that these isoforms can be resolved by capillary one electrophoresis (CZE) using non-treated capillaries. CZE analyses can be performed on as little as 5ng of protein. Needle-like crystals of natural fibrolase were harvested and their composition analyzed by CZE which revealed the same ratio of isoforms in the crystals as was found in the sample of natural enzyme. Recombinant fibrolase was shown to contain at least two isoforms which could be resolved by cation-exchange HPLC. Analysis by CZE revealed that the early eluting fraction from cation-exchange HPLC contained only one isoform and this isoform corresponded to one of the isoforms of natural fibrolase. The late eluting fraction contained two components when analyzed by CZE.
CZE has been shown to be a powerful tool for the analysis of protein isoforms and is useful in assessing the interactions between proteins and metal ions.
CZE and enzymatic activity measurements were used to assess the effects of metal replacement on fibrolase following EDTA titration. To stabilize the enzyme, 4M urea was added to the buffers used. This minimized intermoleeular interactions which appeared to occur after removal of zinc. It was shown that a 30 minute incubation in 10m.M EDTA completely removed zinc from the enzyme.