Monitoring of Dithiothreitol Clearance by Micellar Electrokinetic Chromatography
| Authors | |
|---|---|
| Year of publication | 2001 |
| Type | Article in Proceedings |
| Conference | Book of abstract 2nd International symposium "Separations in BioSciences SBS 2001" |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | DTT; On-column reaction |
| Description | Dithiothreitol (DTT) is a well known sulfhydryl reagent used to protect the sulfhydryl groups of small molecular compounds and proteins in a variety of biochemical applications. The radioprotective action of the compound has received significant attention for a number of years. Recently DTT has been applied in studies on heat cell killing as a result of interest in hyperthermia as a cancer treatment modality. DTT is also routinely used to solubilize recombinant proteins from inclusion bodies. Previous methods used to determine DTT in solutions have included spectrophotometry and HPLC with UV spectrophotometric and fluorescence detection. We proposed the application of the on-column reaction of DTT with the reactive disulphide - 2,2-dipyridyldisulphide (DPDS) for monitoring of DTT clearance. DPDS and a sample of DTT are injected consecutively in the capillary as two discrete plugs separated with short plug of background electrolyte. Due to the differences in the mobilities of the DPDS and DTT plugs on column mixing and reaction occur. DTT is thus quantitatively transformed into a mixed disulphide concomitantly with formation of equimolar amounts of the 2-thiopyridone that is separated by micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of DTT is thus estimated indirectly from the result of 2-thiopyridone determination. |
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