Sulfhydryl Residues Thiolation

Modification of Amines with 2-lminothiolane (Traut s Reagent) 

At high pH (10.0), Traut s reagent also is reactive with aliphatic and aromatic hydroxyl groups, although the rate of reaction with these groups is only about 0.01 that of primary 

Proteins modified with 2-iminothiolane are subject to disulfide formation upon sulfhydryl oxidation. This can cause unwanted conjugation, potentially precipitating the protein. The addition of a metal-chelating agent such as EDTA (0.01-0.1M) will prevent metal-catalyzed oxidation and maintain sulfhydryl stability. In the presence of some serum proteins (i.e., BSA) a 0.1M concentration of EDTA may be necessary to prevent metal-catalyzed oxidation, presumably due to the high contamination of iron from hemolyzed blood. 

Dissolve the Traut s reagent (Thermo Fisher) in water at a concentration of 2mg/ml (makes a 14.5 mM stock solution). The solution should be used immediately. For the modification of IgG at a concentration of lOmg/ml using a 10-fold molar excess of Traut s reagent, add 45.8 pi of the stock solution to each ml of the protein solution. 

React for 1 hour at room temperature (a 4°C reaction temperature may be used successfully as well). 

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Thus, this reagent can be used to label fluorescently proteins and other biomolecules containing free sulfhydryl residues. If there are no —SH groups available, their creation can be accomplished by reduction of indigenous disulfides or through the use of various thiolation reagents (Chapter 1, Section 4.1). 

The amine groups on these fragments also may be modified with thiolating agents, such as SATA or 2-iminothiolane, to create sulfhydryl residues suitable for coupling to maleimide-activated enzymes (Section 1.1, this chapter) (Figure 20.13). Amine groups further may be utilized... 

For instance, if toxin A chain—antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of excess cross-linker, the activated antibody can be directly mixed with isolated A chain to create the conjugate (Fig. 320). This procedure makes use of the indigenous sulfhydryl residues produced during reductive separation of the A and B chains and therefore does not require cross-linker thiolation of one of the proteins. 

Oligonucleotide probes that have been modified with an amine-terminal spacer arm using any of the methods discussed in Sections 1 and 2, may be thiolated to contain a sulfhydryl residue. Theoretically, any of the amine-reactive thiolation reagents described in Chapter 1, Section 4.1, may be used to convert an amino group on a DNA... 

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