TsT-mPEG

Remove excess solvents by rotary evaporation. The TsT-mPEG should be used immediately or stored in anhydrous conditions at 4°C. 

Dissolve the protein to be modified with TsT-mPEG in ice-cold 0.1 sodium borate, pH 9.4, at a concentration of 2-10 mg/ml. Other buffers at lower pH values (down to pH 7.2) can be used and still obtain modification, but the yield will be less. Avoid amine-containing buffers such as Tris or the presence of sulfhydryl-containing compounds, such as disulfide reductants. 

Slowly add TsT-mPEG to the protein solution at a level of at least a 5-fold molar excess over the desired modification level. For example, Gotoh et al. (1993) added 100 lmg of TsT-mPEG-5000 to 19 mg of protein dissolved in 6 ml of buffer. Add the polymer over a period of about 15 minutes with stirring at 4°C. 

Remove excess TsT-mPEG by dialysis or gel filtration using a column of Sephacryl S-300. 

The following protocol for mPEG activation using TsT and its coupling to proteins is based on the protocols of Abuchowski et al. (1977b) and Gotoh et al. (1993). 

Add to the TsT solution, 50 g of mPEG-5000 (monomethoxypolyethylene glycol having a molecular weight of 5000). Mix well to dissolve. 

The three reactive chlorines on TsT have dramatically different reactivities toward nucleophiles in aqueous solution. The first chlorine is reactive toward hydroxyls as well as primary and secondary amine groups at 4°C and a pH of 9 (Mumtaz and Bachhawat, 1991 Abuchowski et al., 1977a). Once the first chlorine is coupled, the second one requires at least room temperature conditions at the same pH to efficiently react. If two chlorines are conjugated to nucleophilic groups, the third is even more difficult to couple, requiring at least 80°C. After activation of mPEG with TsT, it is therefore, for all practical purposes, only possible to couple one additional component to the triazine ring. 

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