Carboxylates or Phosphates with Cystamine

The carbodiimide of choice used to couple cystamine to carboxylate- or phosphate-containing molecules is most often the water-soluble carbodiimide, EDC hydrochloride Chapter 3, Section 1.1). This reagent rapidly reacts with carboxylates or phosphates to form an active ester intermediate, which is highly reactive toward primary amines. The reaction is efficient from pH 4.7 to 7.5, and a variety of buffers may be used, providing they don t contain competing groups. 

Using this approach, EGF has been successfully conjugated by disulfide exchange to the A chain of diphtheria toxin (Shimisu et al., 1980). A cystaminyl derivative of insulin also could be conjugated to the A chain of diphtheria toxin by this method (Miskimins and Shimizu, 1979). Other references to disulfide exchange using cystamine include Oeltmann and Forbes (1981) and Bacha et al. (1983) who prepared antibody-toxin and peptide-toxin conjugates, respectively. 

Using this approach, EGF has been successfully conjugated by disulfide exchange to 

The following protocol is useful for the modification erf proteins with cystamine with subsequent reduction to create the free sulfhydryl. 

Dissolve cystamine (Aldrich) in the reaction buffer at a concentration of 2.25 mg/ml (10 mM). Add an aliquot of this solution to the protein solution to be modified. Use about a 10- to 20-fold molar excess of cystamine over the amount of protein present. For a protein of MW 100,000 at a concentration of 10 mg/ml, add 10 jl1 of the stock cystamine solution to each milliliter of protein solution to obtain a 10-fold molar excess. 

Add EDC (Pierce) to the solution prepared in (2) to obtain at least a five-fold molar excess over the amount of cystamine present. React for 2 h at room temperature. 

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