Lowering the pH to 4.5 and using TCEP (5-300 mM) as the reductant revealed the presence of minor components along the unfolding pathway of kalata B1.
Kalata B1 (0.5Â¬mg/ml) was dissolved in 0.1Â¬M ammonium bicarbonate, pH 8.5,Â¬containing varying concentrations of dithiothreitol (DTT) or TCEP (0.5-300 mM). To monitor the unfolding, aliquots were removed at various time intervals and quenched with 4% aqueous trifluoroacetic acid. Samples were analyzed using RP-HPLC and LCMS. A large-scale reduction was performed for the subsequent oxidative refolding experiments. Kalata B1 was reduced with an excess of DTT (1Â¬M) in ammonium bicarbonate (pH 8.5) at room temperature for 2Â¬h. The reduced material was purified on semi-preparative RP-HPLC at 3Â¬ml/min.
The reductive unfolding of kalata B1 was examined using both DTT and TCEP as reducing agents in the presence and absence of isopropyl alcohol. Isopropyl alcohol did not appear to influence the reactions. The reactions performed at various concentrations of DTT (5-100 mM) at pH 8.5Â¬essentially show only reduced and fully oxidized species with no significant accumulation of any partially reduced species. However, lowering the pH to 4.5Â¬and using TCEP (5-300 mM) as the reductant revealed the presence of minor components along the unfolding pathway. LCMS analysis allowed determination of the mass of the two most abundant intermediates, which correspond to a one-disulfide (Ia) and a two-disulfide (IIb) species. A very minor peak was consistently observed in the reductive unfolding at the retention time observed for intermediate IIa. Isolation of this peak from RP-HPLC and mass spectrometry confirmed that the mass was consistent with intermediate IIa.