Barrington P L, Condrea E, Soons K R, Yang C C, Rosenberg P
Toxicon. 1984;22(5):743-58. doi: 10.1016/0041-0101(84)90157-0.
By treating Naja nigricollis and Naja naja atra phospholipase A2 with carbodiimide and semicarbazide, we obtained derivatives having varied numbers of modified carboxylate groups. When tested on artificial and natural substrates, derivatives of both enzymes with a modified carboxylate group at the active site (Asp-49) retained little enzymatic activity (1/41 to 10%). However, the derivatives of N. nigricollis also lost most of their lethal potency (5% of native), while those of N. n. atra retained considerable lethality (29%). Carboxyl modification with protection of Asp-49 in N. n. atra enzyme resulted in a derivative with lethal potency equal to or greater than the native enzyme and enzymatic activity which was low on all substrates (12-17% of native). Similar protection of Asp-49 at the active site in N. nigricollis enzyme produced a derivative with decreased enzymatic activity on artificial substrate (22% of native) and decreased lethality (17-33% of native), but with full enzymatic activity on natural substrates. When tested on electrical and mechanical properties of the isolated perfused heart and the isolated ventricle muscle wall, the derivatives of both enzymes retained considerably more of the cardiotoxic activity than would have been expected based on their residual enzymatic activity. The one exception occurred with the least modified N. nigricollis derivative which had an unaltered Asp-49, this enzyme retained both cardiotoxic activity and full enzymatic activity on natural substrates. The extent of phospholipid hydrolysis following treatment was measured in the isolated heart preparation and in hearts removed from mice following i.v. injection of the phospholipases. Very low levels of phospholipid hydrolysis were observed and no correlation could be made between the extent of hydrolysis and the pharmacological potencies of these enzymes. Modification of the enzymatic active site, whether of Asp-49 in this study of His-48 in prior studies, leads to a large decrease in both enzymatic activity and lethal potency. Asp and Glu residues outside of the enzymatic site contribute significantly to the lethal potency of the N. nigricollis enzyme and to the enzymatic activity of the N. n. atra enzyme. Based on these and previous data we conclude that changes in isoelectric points are not responsible for altered lethal potencies following chemical modification and that some pharmacological effects of snake venom phospholipases A2 are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.
通过用碳二亚胺和氨基脲处理黑颈眼镜蛇和中华眼镜蛇的磷脂酶A2，我们得到了具有不同数量修饰羧基的衍生物。在人工和天然底物上进行测试时，两种酶在活性位点（Asp-49）带有修饰羧基的衍生物保留的酶活性很低（1/41至10%）。然而，黑颈眼镜蛇的衍生物也失去了大部分致死效力（为天然酶的5%），而中华眼镜蛇的衍生物保留了相当高的致死性（29%）。对中华眼镜蛇酶中的Asp-49进行羧基修饰并加以保护，得到的衍生物致死效力等于或高于天然酶，且在所有底物上的酶活性都很低（为天然酶的12 - 17%）。对黑颈眼镜蛇酶活性位点的Asp-49进行类似保护，产生的衍生物在人工底物上酶活性降低（为天然酶的22%），致死性降低（为天然酶的17 - 33%），但在天然底物上具有完整的酶活性。在对离体灌注心脏和离体心室肌壁的电和机械特性进行测试时，两种酶的衍生物保留的心脏毒性活性比根据其残余酶活性预期的要高得多。唯一的例外是修饰最少的黑颈眼镜蛇衍生物，其Asp-49未改变，该酶在天然底物上既保留了心脏毒性活性又保留了完整的酶活性。在离体心脏制剂以及静脉注射磷脂酶后从小鼠身上取出的心脏中，测量了处理后磷脂水解的程度。观察到磷脂水解水平非常低，并且水解程度与这些酶的药理效力之间没有相关性。酶活性位点的修饰，无论是本研究中的Asp-49还是先前研究中的His-48，都会导致酶活性和致死效力大幅降低。酶活性位点之外的Asp和Glu残基对黑颈眼镜蛇酶的致死效力以及中华眼镜蛇酶的酶活性有显著贡献。基于这些及先前的数据，我们得出结论，等电点的变化并非化学修饰后致死效力改变的原因，并且蛇毒磷脂酶A2的一些药理作用是由于非酶作用，这表明存在两个不同但可能重叠的活性位点。
