A new heterobifunctional cross-linking reagent for the study of biological interactions between proteins. I. Design, synthesis, and characterization.

A new heterobifunctional photoaffinity probe, N-(4-azidobenzoylglycyl)-S-(2-thiopyridyl)-cysteine (AGTC) can be synthesized from cysteine via a coupling of the N-hydroxysuccinimide ester of 4-azidobenzoylglycine to S-(2-thiopyridyl) cysteine. AGTC is readily dissolved in an aqueous buffer at pH 7.5 and is stable at room temperature in aqueous media ranging in pH from 3.0 to 9.0. No degradation is observed when AGTC in aqueous solution at pH 7.5 is exposed to laboratory fluorescent light at room temperature for 1 week. The disulfide bridge moiety of AGTC is stable to the conditions of photolysis used to activate the arylazido group for cross-linking. AGTC is incorporated (over 90% within 2 h) into such proteins as rabbit skeletal troponin C, alpha-tropomyosin, and actin through disulfide bridge formation. The degree of incorporation of AGTC into proteins can be monitored by spectrophotometric determination of the release of pyridine-2-thionine at 343 nm. This disappearance of the azido moiety of AGTC or the photoaffinity-labeled tropomyosin (AGC-tropomyosin) during photolysis can be followed by the decrease in the absorbance at 270 nm. When AGC-tropomyosin was treated with a 6 M urea, 0.1 M dithiothreitol buffer overnight, 92% of the photoaffinity probe was removed from the sulfhydryl group of tropomyosin. This reduced tropomyosin can be relabeled (over 86%) with a freshly prepared solution of AGTC, indicating the reversibility of the disulfide attachment.