CHEMICAL PROBES OF EXTENDED BIOLOGICAL STRUCTURES - SYNTHESIS AND PROPERTIES OF CLEAVABLE PROTEIN CROSS-LINKING REAGENT [DITHIOBIS(SUCCINIMIDYL-S-35 PROPIONATE)
CHEMICAL PROBES OF EXTENDED BIOLOGICAL STRUCTURES - SYNTHESIS AND PROPERTIES OF CLEAVABLE PROTEIN CROSS-LINKING REAGENT [DITHIOBIS(SUCCINIMIDYL-S-35 PROPIONATE)
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DOI：
10.1016/0022-2836(76)90011-5
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发表时间：
1976-01-01
影响因子：
5.6
通讯作者：
FAIRBANKS, G
中科院分区：
文献类型：
--
作者：
LOMANT, AJ;FAIRBANKS, G
The synthesis and properties of a new cleavable protein cross-linking reagent, [35S]dithiobis(succinimidyl propionate), are detailed. Free primary and secondary aliphatic amino groups are quantitatively acylated by the reagent in either organic or aqueous media within 2 min at 23.degree. C. The half-time for hydrolysis of the active ester termini in buffer at pH 7 is 4-5 h, so that protein cross-linkage can be optimized by application of low concentrations of reagent. Accessible amino groups of bovine Hb are acylated with extreme rapidity of 0.degree. C in pH 7 buffer when [35S]dithiobis(succinimidyl propionate) is applied in 0.4 to 9-fold molar excess. Submicrogram quantities of the cross-linked Hb subunits which result are detectable by monitoring the 35S distribution in sodium dodecyl sulfate-polyacrylamide gels. In addition to amine acylation, 2 of the 6 thiol groups in Hb, tentatively located at cysteine 93 of the .beta. chains, are reversibly modified at 0.degree. C by mercaptan-disulfide interchange with the reagent or its bis amide analogs. This equilibrium-controlled, pH-dependent reaction occurs at a slower rate than acylation, and is blocked by short preincubation of the protein with N-ethylmaleimide or by addition of 3,3''-dithiodipropionamide (or other disulfides) to the reaction mixture. Disulfides introduced into Hb by acylation and interchange are quantitatively cleaved by reduction for 30 min at 37.degree. C with 10 mM-dithioerythritol buffered at pH 8.5. The properties of high reactivity under mild conditions, long solution half-life, and the radioactive label make (35S]dithiobis(succinimidyl propionate) a particularly useful and versatile probe of extended structures in a variety of biological systems.
