1
edit
Thiols and disulfides: Difference between revisions
→Method according to Thannhauser: determination of disulfides and thiols
No edit summary |
|||
| Line 24: | Line 24: | ||
NTSB "synthesis": | NTSB "synthesis": | ||
29.8 mg of DTNB is dissolved in 3 ml 1 M Na<sub>2</sub>SO<sub>3</sub>. Adjust pH to 9-9.5. The DTNB is cleaved by the sulfite as indicated by the intense yellow color formed. The products are NTSB (nitro thio | 29.8 mg of DTNB is dissolved in 3 ml 1 M Na<sub>2</sub>SO<sub>3</sub>. Adjust pH to 9-9.5. The DTNB is cleaved by the sulfite as indicated by the intense yellow color formed. The products are NTSB (2-nitro 5-thio sulfo benzoic acid) and NTB. The NTB reoxidizes with oxygen to DTNB which is subsequently cleaved again to NTSB and NTB. The progress of the conversion of DTNB into NTSB can be easily followed by decrease in 412 nm or just by the naked eye by decrease in yellow color. The residual solution is pale yellow. The final NTSB solution is 50 mM and is stable for at least 6 months at -20°C. | ||
| Line 33: | Line 33: | ||
* 5. Dilute protein in 1ml reaction buffer at a final concentration of 10-40 µM disulfide. The disulfide is cleaved into a thiol and a thio-sulfonate. Thiols are determined by NTSB. Add 10 µl of NTSB stock as prepared above. Read absorption at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol or disulfide. | * 5. Dilute protein in 1ml reaction buffer at a final concentration of 10-40 µM disulfide. The disulfide is cleaved into a thiol and a thio-sulfonate. Thiols are determined by NTSB. Add 10 µl of NTSB stock as prepared above. Read absorption at 412 nm. Extinction coefficient is 13,600 M-1 cm-1 per thiol or disulfide. | ||
* 6. Subtraction of number of determined thiols yields number of disulfides | * 6. Subtraction of number of determined thiols yields number of disulfides | ||
=== References === | === References === | ||