Wilbur D S, Pathare P M, Hamlin D K, Weerawarna S A
Department of Radiation Oncology, University of Washington, Seattle 98195, USA.
Bioconjug Chem. 1997 Nov-Dec;8(6):819-32. doi: 10.1021/bc970053e.
Polymerization and/or cross-linking of recombinant streptavidin (r-SAv) with biotin derivatives containing two biotin moieties (biotin dimers) or three biotin moieties (biotin trimers) has been investigated as a model for reagents to be used to increase the amount of radioactivity on cancer cells in tumor pretargeting protocols. In the investigation, six biotin dimers and three biotin trimers were synthesized. Most biotin derivatives synthesized had ether containing linker molecules incorporated to improve their aqueous solubility. The synthesized biotin dimers contained linker moieties which provided distances (when fully extended) of 13-49 A between biotin carboxylate carbon atoms, and the biotin trimers contained linker moieties which provided distances of 31-53 A between any two biotin carboxylate atoms. All of the biotin derivatives were evaluated for their ability to polymerize r-SAv in solution. When the biotin derivatives were mixed with r-SAv, none of the biotin dimers caused polymerization, but all of the biotin trimers resulted in complete polymerization. Some of the biotin dimers did cross-link r-SAv (to form r-SAv dimers, trimers, etc.), but the percentage of cross-linking was low (< or = 40%). The length of the linker molecule was important in cross-linking of biotin dimers. While linkers which provided distances of 13 and 19 A between biotin carboxylate carbon atoms did not result in cross-linking, a linker which provided a 17 A distance resulted in a small (< or = 10%) amount of cross-linking. Also cross-linking was increased in biotin dimers with linkers which provided distances between biotin carboxylate carbon atoms of > or = 23 A. Cross-linking of streptavidin bound in polystyrene wells with biotin dimers and trimers was also examined. In those experiments, an excess of each biotin derivative was incubated at 37 degrees C for 10-30 min in polystyrene wells containing bound SAv. After the excess biotin derivative was rinsed from the wells, an excess of r-[125I]SAv was incubated for another 10-30 min. The amount of r-[125I]SAv bound after rinsing the excess from the wells was an indicator of the extent of cross-linking that occurred. The process of alternating additions of reagents was repeated four times to demonstrate that bound radioactivity could be increased with each addition of [125I]SAv. The results of cross-linking r-SAv in polystyrene wells paralleled results from cross-linking in solution.
重组抗生物素蛋白（r-SAv）与含有两个生物素部分（生物素二聚体）或三个生物素部分（生物素三聚体）的生物素衍生物的聚合和/或交联已作为一种模型进行研究，该模型用于肿瘤预靶向方案中增加癌细胞上放射性剂量的试剂。在该研究中，合成了六种生物素二聚体和三种生物素三聚体。合成的大多数生物素衍生物都引入了含醚连接分子以改善其水溶性。合成的生物素二聚体包含连接部分，其在生物素羧酸盐碳原子之间提供的距离（完全伸展时）为13 - 49埃，生物素三聚体包含连接部分，其在任意两个生物素羧酸盐原子之间提供的距离为31 - 53埃。评估了所有生物素衍生物在溶液中使r-SAv聚合的能力。当生物素衍生物与r-SAv混合时，没有一种生物素二聚体引起聚合，但所有生物素三聚体都导致完全聚合。一些生物素二聚体确实使r-SAv交联（形成r-SAv二聚体、三聚体等），但交联百分比很低（≤40%）。连接分子的长度在生物素二聚体的交联中很重要。虽然在生物素羧酸盐碳原子之间提供13和19埃距离的连接子不会导致交联，但提供17埃距离的连接子会导致少量（≤10%）交联。在生物素羧酸盐碳原子之间提供≥23埃距离的连接子的生物素二聚体中，交联也会增加。还研究了聚苯乙烯孔中结合的抗生物素蛋白与生物素二聚体和三聚体的交联。在那些实验中，将每种生物素衍生物的过量物在37℃下于含有结合的SAv的聚苯乙烯孔中孵育10 - 30分钟。从孔中冲洗掉过量的生物素衍生物后，再将过量的r-[125I]SAv孵育10 - 30分钟。从孔中冲洗掉过量物后结合的r-[125I]SAv的量是发生交联程度的指标。重复试剂交替添加过程四次，以证明每次添加[125I]SAv时结合的放射性都可以增加。聚苯乙烯孔中r-SAv的交联结果与溶液中的交联结果相似。
