Cystamine

Cystamine dihydrochloride, S,5-(L,L)-Cystathionine, Cysteamine, Cysteamine hydrochloride, ( )-Cysteic acid, S-Cysteic acid (H2O), L-Cysteine hydrochloride (H2O), ( )-Cysteine hydrochloride and L-Cystine, Cytidine, see entries in Chapter 6. 

To the methods reviewed in an earlier volume 1 may be added the preparation by the oxidation of cystamine 2 and by the decarboxylation of cysteic acid.3 The method given in the procedure has appeared recently in the literature.4... 

Rhena-/3-ketoimine derivatives of several selected 2-ethylamino compounds have been prepared (36). These amines include 2-chloroethyl-amine (a DNA-alkylating reagent), cystamine ( a heparin antagonist), histamine (a potent vasodilator), tryptamine and O-methylserotonin (two indole alkaloids), and 0,0-dimethyldopamine (an adrenergic drug derivative). 

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. 

Figure 1.71 Cystamine may be used to label protein carboxylate groups using the water-soluble carbodiimide EDC.

Figure 1.72 Cystamine may be used to label phosphate groups, such as at the 5 -end of oligonucleotides, via a carbodiimide reaction using EDC. The resultant phosphoramidate linkage is a common way to modify oligonucleotides at the 5 -end.

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.

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. 

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). 

Modification of Nucleic Acids and Oligonucleotides with Cystamine... 

DNA or RNA also may be modified with cystamine at the 5 -phosphate group using a carbodiimide reaction. See Chapter 27, Section 2.2 for a complete discussion of the labeling protocol. 

Mercapto- Cystamine Lipoic acid Dihydrolipoic DSP Thioacetic... 

Figure 21.10 Cystamine may be used to make immunotoxin conjugates by a disulfide interchange reaction. Modification of antibody molecules using an EDC-mediated reaction creates a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol can be coupled to the cystamine-modified antibody to form disulfide crosslinks.

The following protocol describes the modification of DNA or RNA probes at their 5 -phosphate ends with a bis-hydrazide compound, such as adipic acid dihydrazide or carbohydrazide. A similar procedure for coupling the diamine compound cystamine can be found in Section 2.2 (this chapter). 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

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