A 125I-radiolabel transfer crosslinking reagent with a novel cleavable group.
A 125I-radiolabel transfer crosslinking reagent with a novel cleavable group.
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具有新型可裂解基团的 125I-放射性标记转移交联试剂。
DOI：
10.1016/0003-2697(85)90487-7
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发表时间：
1985
影响因子：
2.9
通讯作者：
Schwartz,MA
中科院分区：
文献类型：
--
作者：
Schwartz,MA
研制了一种新型化学交联试剂1-[N-（2-羟基-5-叠氮苯甲酰基）-2-氨基乙基]-4-（N-羟基琥珀酰亚基）-琥珀酸盐（HAHS）。它分三步合成，作为未标记的前体储存，然后在使用前立即碘化。该试剂的一端有琥珀酰亚胺酯，因此它可以与纯化的蛋白质共价连接，另一端有放射性碘化的苯基叠氮基团，因此在光解时，它可以与附近的分子形成交联。16-Å连接区含有一个酯基，该酯基在光解前在中性pH下非常稳定，但在碱中水解约1 min，光解后自发水解。因此，酯的光解和裂解导致放射性标记从初始蛋白质转移到它的邻居。125i - hahs -protein A与IgG孵育、光解和裂解后，27%的标记转移到IgG重链上。这种转移被过量的未标记蛋白A所消除，并被低浓度的DTT所猝灭。转移到不相互作用的蛋白质上的标签量要少得多。当125i - hahs -spectrin结合到spectrin耗尽的红细胞膜上，进行光解和切割时，标签仅转移到锚蛋白和能带3上。这种转移被过量的未标记的幽灵蛋白阻断，并被阻止幽灵蛋白结合的条件大大减少。
A new chemical crosslinking reagent, 1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate, or HAHS, has been developed. It is synthesized in three steps and stored as an unlabeled precursor, and then iodinated immediately before use. The reagent has a succinimide ester at one end so that it can be covalently attached to a purified protein, and a radioiodinated phenyl azide group at the other end, so that upon photolysis it can form crosslinks to nearby molecules. The 16-Å connecting region contains an ester group which is very stable at neutral pH before photolysis, but which hydrolyzes in about 1 min in base, and hydrolyzes spontaneously after photolysis. Thus, photolysis and cleavage of the ester result in transfer of the radiolabel from the initial protein to its neighbors. When125I-HAHS-protein A was incubated with IgG, photolyzed, and cleaved, 27% of the label was transferred to the IgG heavy chain. This transfer was abolished by an excess of unlabeled protein A, and was quenched by low concentrations of DTT. Much lower amounts of label were transferred to noninteracting proteins. When125I-HAHS-spectrin was bound to spectrin-depleted red blood cell membranes, photolyzed, and cleaved, label was transferred only to ankyrin and to band 3. This transfer was blocked by excess unlabeled spectrin and was greatly diminished by conditions which prevent binding of spectrin.