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Synthesis of 3,3'-bis(sulfonato)-4,4'-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity.

Nature protocols (2007-04-05)
Darcy C Burns, Fuzhong Zhang, G Andrew Woolley
ABSTRACT

This protocol describes a procedure for the synthesis of 3,3'-bis(sulfonato)-4,4'-bis(chloroacetamido)azobenzene (BSBCA), a water-soluble, thiol-reactive, photo-switchable cross-linker. In addition, a protocol is outlined for installing the cross-linker in an intramolecular fashion onto proteins bearing two surface-exposed Cys residues. BSBCA is designed to be used as an in vitro activity switch that operates by exerting temporal and reversible photo-control over alpha-helix content within synthetic peptides and recombinant proteins. Synthesis of the cross-linker requires approximately 4.5 d, and cross-linking can be performed in 10-12 h.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Chloroacetic anhydride, 95%
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Acetic anhydride, ACS reagent, ≥98.0%
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Sodium hypochlorite solution, reagent grade, available chlorine 4.00-4.99 %
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Sodium hypochlorite solution, reagent grade, available chlorine 10-15 %
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Sodium hydroxide, ACS reagent, ≥97.0%, pellets