Trikha M, Schmitmeier S, Markland F S
Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Los Angeles 90033.
Toxicon. 1994 Dec;32(12):1521-31. doi: 10.1016/0041-0101(94)90310-7.
Fibrolase, a zinc metalloproteinase possessing direct-acting fibrinolytic activity, has been previously purified from southern copperhead (Agkistrodon contortrix contortix) snake venom. We recently reported that a pool of southern copperhead venom from different geographical locations possesses two isoforms of fibrolase (fib1 and fib2) [Loayza, S. L. et al. (1994) J. Chromat. B, in press]. We now report that venom from individual southern copperhead snakes contains the two isoforms which can be separated by a three-step high performance liquid chromatography (HPLC) procedure consisting of hydrophobic interaction chromatography, hydroxylapatite chromatography and weak cation exchange chromatography. Utilizing mass spectrometry we determined that fib1 has a molecular mass of 22,879 atomic mass units (amu) compared to 22,753 amu for fib2. These results support earlier observations during amino acid sequence analysis that a truncated version of the enzyme is produced which is missing the amino-terminal amino acid (< Glu-Arg-Phe-Pro vs. the intact enzyme < Glu-Gln-Arg-Phe-Pro, where < Glu is cyclized glutamine). The truncated version of fibrolase (fib2) has full fibrinolytic activity compared to fib1. EC50 values (concentration of enzyme required to degrade 50% of fibrin in a micro-fibrin plate assay) are 6.4 (+/- 1.0) microM and 5.2 (+/- 0.8) microM for fib 1 and fib2, respectively. Therefore, loss of the amino-terminal amino acid does not appear to influence enzymatic activity. We conclude that the two isoforms of fibrolase arise from variations in the molecular processing of the enzyme by the snake venom gland rather than being caused by the pooling of southern copperhead venoms from different geographical locations.
纤维溶酶是一种具有直接纤溶活性的锌金属蛋白酶，此前已从南方铜头蝮蛇（Agkistrodon contortrix contortix）毒液中纯化得到。我们最近报道，来自不同地理位置的南方铜头蝮蛇毒液池含有两种纤维溶酶同工型（fib1和fib2）[洛亚萨，S. L. 等人（1994年）《色谱杂志B》，即将发表]。我们现在报道，单个南方铜头蝮蛇的毒液含有这两种同工型，可通过由疏水相互作用色谱、羟基磷灰石色谱和弱阳离子交换色谱组成的三步高效液相色谱（HPLC）程序进行分离。利用质谱法我们确定，fib1的分子量为22,879原子质量单位（amu），而fib2为22,753 amu。这些结果支持了早期氨基酸序列分析中的观察结果，即产生了该酶的截短版本，其缺少氨基末端氨基酸（<Glu-Arg-Phe-Pro，而完整酶为<Glu-Gln-Arg-Phe-Pro，其中<Glu为环化谷氨酰胺）。与fib1相比，纤维溶酶的截短版本（fib2）具有完全的纤溶活性。在微纤维平板试验中，fib1和fib2的EC50值（降解50%纤维蛋白所需的酶浓度）分别为6.4（±1.0）μM和5.2（±0.8）μM。因此，氨基末端氨基酸的缺失似乎不影响酶活性。我们得出结论，纤维溶酶的两种同工型源于蛇毒腺对该酶分子加工的差异，而非不同地理位置的南方铜头蝮蛇毒液混合所致。
