Proteins with Cystamine

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

Dissolve cystamine (Aldrich) in the reaction buffer at a concentration of 2.25mg/ml (lOmM). 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 lOmg/ml, add 10pi of the stock cystamine solution to each ml of protein solution to obtain a 10-fold molar excess. 

Add EDC (Thermo Fisher) to the solution prepared in (2) to obtain at least a 5-fold molar excess over the amount of cystamine present. React for 2 hours at room temperature. 

Separate excess cystamine and EDC (and reaction by-products) from the modified protein by dialysis or gel filtration using 10 mM sodium phosphate, 0.15M NaCl, pH 7.2. A desalting column may be used for the gel filtration procedure (i.e., Zeba spin columns from Thermo Fisher). 

To reduce the disulfide groups, add DTT at a concentration of 0.5mg DTT per mg of modified protein. A stock solution of DTT may be prepared to make it easier to add it to a small amount of protein solution. In this case, dissolve 20 mg of DTT per ml of 0.1 M sodium acetate, 0.1 M NaCl, pH 4.5. Add 25 pi of this solution per mg of modified protein. 

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Figure 1.73 The disulfide group of a cystamine-modified protein may undergo disulfide interchange reactions with another sulfhydryl-containing protein to yield a disulfide-linked conjugate.

The first natural sulfhydryl activator of FDPase to be described was cystamine, which at pH 7.5 was found to undergo a disulfide exchange reaction with 2 reactive cysteine residues in the protein (45), leading... 

Fig. 1.5. Dependence of the electroenz3maatic response (Ay) of lactate dehydrogenase modified gold electrodes on the time of protein layer growth (a) mixed 3-mercaptopropamol and 3-mercaptopropionic acid SAM derivatized with 1,4-diamino-butane ligand fi-ee- ( ), CB- (O) and Ll-anchored (A) (b) cystamine SAM derivatized with fumaric acid ligand fi-ee- ( ), CB- (O) and Ll-anchored (A). The monolayers were incubated in a 0.36mgml enzyme solution in 50 mM Na-phosphate buffer, pH 7.0. Reproduced from [240] with permission.

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