Coordination properties of dithiobutylamine (DTBA), a newly introduced protein disulfide reducing agent.

The acid-base properties and metal-binding abilities of (2S)-2-amino-1,4-dimercaptobutane, otherwise termed dithiobutylamine (DTBA), which is a newly introduced reagent useful for reducing protein and peptide disulfides, were studied in solution using potentiometry, (1)H NMR spectroscopy, spectropolarimetry, and UV-vis spectroscopy. The list of metal ions studied here includes Zn(II), Cd(II), Ni(II), Co(II), and Cu(I). We found that DTBA forms specific and very stable polynuclear and mononuclear complexes with all of these metal ions using both of its sulfur donors. DTBA forms complexes more stable than those of the commonly used disulfide reducing agent DTT, giving it more interference capacity in studies of metal binding in thiol-containing biomolecules. The ability of DTBA to strongly bind metal ions is reflected in its limited properties as a thiol protectant in their presence, which is manifested through slower disulfide reduction kinetics. We found that this effect correlated with the stabilities of the complexes. Additionally, the reducing properties of DTBA toward MMTS-modified papain (MMTS = S-methylmethanethiosulfonate) were also significantly affected by the investigated metal ions. In this case, however, electrostatic interactions and stereospecific effects, rather than metal-binding abilities, were found to be responsible for the reduced protective properties of DTBA. Despite its limitations, a high affinity toward metal ions makes DTBA an attractive agent in competition studies with metalloproteins.