Monitoring of DTT clearence by MEKC
| Authors | |
|---|---|
| Year of publication | 2002 |
| Type | Article in Proceedings |
| Conference | Book of abstracts of 6th Meeting of biochemists and molecular biologists |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | DTT; MEKC |
| Description | Dithiotreitol (DTT) is a well-known sulfhydryl reagent. Since 1964 when Cleland described its function it was used to protect the sulfhydryl groups of small molecular compounds as well as proteins in variety of biochemical applications. The radioprotective action of the compound has received significant attention for a number of years and its mechanisms have been examined in chemical and biological systems. Moreover DTT has been applied in studies on heat cell killing in hyperthermie as a cancer treatment modality. DTT is also routinely used to solubilize recombinant proteins from inclusion bodies. A new method for specific determination of dithiotreitol using micellar electrokinetic chromatography and on-column reaction with reactive disulfide 2,2-dipyridyldisulphide is described. DTT is in this reaction quantitatively transformed into a mixed disulphide concomitantly with formation of equimolar amount of the 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined spectrophotometrically. The concentration of DTT is thus estimated indirectly from the result of 2-thiopyridone determination. Separation has been achieved using the background electrolyte consisting of 50 mM SDS in 50 mM sodium dihydrogen phosphate (pH 7,5). Optimum separation voltage of 28 kV (positive polarity) and the temperature of capillary 20 oC gave the best analysis. The on-column detection reaction was performed by injection: 5 mM DPDS 50 mbar for 20 s, the background electrolyte 50 mbar for 360 s, the sample 50 mbar for 2 s, the background electrolyte 50 mbar for 2 s. Samples were detected using a diode-array detector at 200 nm and 343 nm with a bandwidth 10 nm. The method can be applied for DTT monitoring both in chemical and biological systems. |
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