Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate)

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 two minutes at 23 °C. By contrast, the half-time for hydrolysis of the active ester termini in buffer at pH 7 is four to five hours, so that protein cross-linkage can be optimized by application of low concentrations of reagent. Accessible amino groups of hemoglobin are acylated with extreme rapidity of 0 °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 hemoglobin subunits which result are detectable by monitoring the ³⁵S distribution in sodium dodecyl sulfate-polyacrylamide gels. In addition to amine acylation, two of the six thiol groups in hemoglobin, tentatively located at cysteine 93 of the β chains, are reversibly modified at 0 °C by mercaptan-disul-fide 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 hemoglobin by acylation and interchange are quantitatively cleaved by reduction for 30 minutes at 37 °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.