Traut’s reagent

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... 

Figure 1.61 Traut s reagent can undergo side reactions after the modification of an amine-containing molecule. The terminal thiol group can recyclize to create another iminothiolane derivative that effectively ties up the thiol.

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. 

Purify the thiolated protein from unreacted Traut s reagent by gel filtration using your buffer of choice (i.e., 20mM sodium phosphate, 0.15M NaCl, ImM EDTA, pH 7.2). The addition of EDTA to this buffer helps to prevent oxidation of the sulfhydryl groups and the resultant disulfide formation. After purification, use the thiolated protein immediately... 

The number of sulfhydryls created on the immunoglobulin using thiolation procedures such as this one is more critical to the yield of conjugated enzyme molecules than the molar excess of maleimide-activated enzyme used in the conjugation reaction. Therefore, it is important to use a sufficient excess of Traut s reagent to obtain a sufficient number of available sulfhydryls. 

Add 2-iminothiolane (Thermo Fisher) to this solution to give a molar excess of 20-40 X over the amount of antibody present (MW of Traut s reagent is 137.63). Addition of solid 2-iminothiolane may be done despite the fact that the compound is relatively insoluble in aqueous solution. As the reagent reacts, it will be completely drawn into solution. Alternatively, a stock solution of Traut s may be made in DMF and an aliquot added to the antibody solution (not to exceed 10 percent DMF in the final solution). 

Activation of Proteins with Traut s Reagent Yielding Proteins with Additional Free SH Groups... 

Mix 4 mg of KLH or a respective amount of a glycerol-containing solution of KLH (volume maximal 60 pi) with 190 pi Soln. A. Weigh 0.6 mg Traut s reagent (2-iminothiolane) into an Eppendorf tube and add the carrier protein solution. Shake at RT for 30 min. 

Traut s reagent is fully water-soluble and reacts with primary amines in the pH range 7—10. The cyclic imidothioester is stable to hydrolysis at acid pH values, but its half-life in solution decreases as the pH increases beyond neutrality. However, even at pH 8 in 25 mM triethanolamine the rate of sulfhydryl formation without added primary amine was found to be negligible. On addition of dipeptide amine, the reagent reacted quickly as evidenced by the production of Ellman s reagent color. The rate of reaction also can be followed by 2-iminothiolane s absorbance at 248 nm (Xmax e = 8 840 M 1 cm 1). As the cyclic imidate reacts with amines, its absorbance at... 

M NaCl, pH 7.2, containing 10 mM EDTA as the buffer. To obtain efficient separation between the reduced antibody and excess reductant, the sample size applied to the column should be at a ratio of no more than 5% sample volume to the total column volume. Collect 0.5-ml fractions and monitor for protein at 280 nm. To monitor the separation of the second peak (excess Traut s reagent), the BCA protein assay reagent (Pierce) may be used according to the procedure described in the previous section, protocol step 4. 

Purify the thiolated toxin from unreacted Traut s reagent by gel filtration using... 

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