Hydroxylamine SATA using

Figure 20.13 The thiolation reagent SATA can be used to create sulfhydryl groups on Fab fragments. After deprotection of the acetylated thiol of SATA with hydroxylamine, conjugation with a maleimide-activated enzyme can take place, producing thioether linkages.

Figure 27.8 SATA may be used to modify a 5 -amine derivative of an oligonucleotide, forming a protected sulf-hydryl. Deprotection with hydroxylamine results in generation of a free thiol.

Oligonucleotides containing amine groups introduced by enzymatic or chemical means may be modified with SATA (Chapter 1, Section 4.1) to produce protected sulfhydryl derivatives. The NHS ester end of SATA reacts with a primary amine to form a stable amide bond. After modification, the acetyl protecting group can be removed as needed by treatment with hydroxylamine under mildly alkaline conditions (Fig. 401). The result is terminal sulfhydryl groups that can be used for subsequent labeling with thiol-reactive probes or activated-enzyme derivatives (Kumar and Malhotra, 1992). 

Sulfhydryl groups are introduced using one of two reagents that can be obtained commercially 2-iminothiolane (2-IT) and SATA (Fig. 4). SATA contains a protected sulfhydryl group to confer stability on the molecule. When a free sulfhydryl group is needed, it can be generated by treatment with hydroxylamine. (Fig. 4). 

Therefore, a preliminary study on aliquots of IgG using 2-IT in molar excesses of 10-100 should be performed. When SATA is used the deacetylation of the substituted IgG should be performed with a freshly prepared solution of 1 M hydroxylamine. Sometimes this solution becomes turbid when the pH is brought to 7.5. This is a sign that the reagent is old and should be changed. 

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