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Thiols and disulfides: Difference between revisions
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=== Method according to Thannhauser: determination of disulfides and thiols === | === Method according to Thannhauser: determination of disulfides and thiols === | ||
The amount of disulfides in a protein is assessed by determination of thiols generated through cleavage of disulfides by sulfite. For the measurements a derivative of DTNB has to be prepared: | The amount of disulfides in a protein is assessed by determination of thiols generated through cleavage of disulfides by sulfite. For the measurements a derivative of DTNB has to be prepared: | ||
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 sulfonic bencoic acid) and NTB. The NTB reoxidizes with oxygen to DTNB which is subsequently cleft 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. | 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 sulfonic bencoic acid) and NTB. The NTB reoxidizes with oxygen to DTNB which is subsequently cleft 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. | ||