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Thiols and disulfides: Difference between revisions
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=== Method according to Riddles: determination of thiols with DTNB === | === Method according to Riddles: determination of thiols with DTNB === | ||
Dilute protein in 1 ml of 6 M Guanidine-HCl, 50mM Tris-HCl, pH 8.3, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 10 µl of 10 mM DTNB (Dithionitrobenzoic acid; Ellman’s reagent) in 100 mM Tris-Cl, pH 7.6. Read absorption at 412 nm. Extinction coefficient is 13,600 M<sup>-1 | Dilute protein in 1 ml of 6 M Guanidine-HCl, 50mM Tris-HCl, pH 8.3, 1 mM EDTA to a final concentration of 10-40 µM thiols. Add 10 µl of 10 mM DTNB (Dithionitrobenzoic acid; Ellman’s reagent) in 100 mM Tris-Cl, pH 7.6. Read absorption at 412 nm. Extinction coefficient is 13,600 M<sup>-1 cm<sup>-1 per thiol. | ||
If the thiols in the protein are oxidizing very fast keep the protein at low pH, which will keep the thiol protonated. Only the thiolate is oxidizing very fast. At low pH the assay with DTNB does not work. | If the thiols in the protein are oxidizing very fast keep the protein at low pH, which will keep the thiol protonated. Only the thiolate is oxidizing very fast. At low pH the assay with DTNB does not work. | ||
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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 synthesized: | 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 synthesized: | ||
NTSB synthesis: | NTSB synthesis: | ||
29.8 mg of DTNB is dissolved in 3 ml 1 | 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. | ||
* 1. Determine thiols as described above. | * 1. Determine thiols as described above. | ||
* 2. Prepare a stock of 6.3 M Guanidine-HCl, 1 mM EDTA, 0.2 M Tris-Cl, pH 9.5. | * 2. Prepare a stock of 6.3 M Guanidine-HCl, 1 mM EDTA, 0.2 M Tris-Cl, pH 9.5. | ||
* 3. Prepare a fresh 2 M | * 3. Prepare a fresh 2 M Na<sub>2</sub>SO<sub>3</sub> solution within 1 mM EDTA / water. | ||
* 4. Prepare always freshly the reaction buffer by mixing 20 parts of buffer of point 2. and 1 part of 2 M Na2SO3 solution | * 4. Prepare always freshly the reaction buffer by mixing 20 parts of buffer of point 2. and 1 part of 2 M Na2SO3 solution | ||
* 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. | ||