, Volume 75, Issue 2, pp 219–250 | Cite as

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD)

The variety of DDD-staining methods demonstrated on ehrlich ascites tumor cells
  • G. Nöhammer


2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specifity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhmmer (1978) and calibrated by Nöhammer et al. (1981), was used.


Reaction Time Protein Content Disulfide Thiol Incubation Time 
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  1. Andersson L-O (1966) The heterogeneity of bovine serum albumin. Biochim Biophys Acta 117:115–133Google Scholar
  2. Andersson L-O, Berg G (1969) Hydrolysis of disulfide bonds in weakly alkaline media. Biochim Biophys Acta 192:534–536Google Scholar
  3. Bahr GF (1966) Introduction to the cytochemistry of sulfhydryl and disulfide groups. In Wied GL (ed) Introduction to quantitative cytochemistry. Academic Press, New YorkGoogle Scholar
  4. Barnard EA (1961) Acylation and diazonium coupling in protein cytochemistry with special reference to the benzoylation-tetrazonium method. In: Danielli JF (ed) General cytochemical methods. Academic Press, New YorkGoogle Scholar
  5. Barrnett JR, Seligman AM (1952) Histochemical demonstration of protein-bound SH-groups. Science 116:323–327Google Scholar
  6. Barrnett JR (1953) Histochemical distribution of protein sulfhydryl groups. J Natl Cancer Inst 13:905–925Google Scholar
  7. Barrnett JR, Seligman AM (1954) Histochemical demonstration of sulfhydryl and disulfide groups of protein. J Natl Cancer Inst 14:769–792Google Scholar
  8. Boross L (1969) Secondary SH-disulfide exchange reactions following the modification of nucleophilic SH-groups in D-glyceraldehyde-3-phosphate dehydrogenase with DTNB (5,5′-dithiobis-(2-nitrobenzoate)). Acta Biochim Biophys Acad Sci Hung 4:57–69Google Scholar
  9. Brocklehurst K, Kierstan M, Little G (1972) The reaction of papain with Ellman's reagent (5,5′-dithiobis-(2-nitro-benzoate)dianion). Biochem J 128:811–816Google Scholar
  10. Brocklehurst K, Little G (1973) Reactions of papain and of low-molecular-weight thiols with some aromatic disulfides: 2,2′-dipyridyl disulfide as a conventient active-site titrant for papain even in the presence of other thiols. Biochem J 133:67–80Google Scholar
  11. Carmichael DF, Keefe M, Pace M, Dixon JE (1979) Formation of interchain disulfide bonds in Bence Jones proteins and Fab(t) fragments of immunoglobulin G through thiol-disulfide interchange. J Biochem 84:1475–1483Google Scholar
  12. Cecil R, Loening UE (1960) The reaction of disulfide groups of insulin with sodium sulphite. Biochem J 76:146–155Google Scholar
  13. Cecil R, Wake RG (1962) The reactions of inter- and intrachain disulfide bonds in proteins with sulphite. Biochem J 82:401–406Google Scholar
  14. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–77Google Scholar
  15. Esterbauer H (1972) Beitrag zum quantitativen histochemischen Nachweis von Sulfhydrylgruppen mit der DDD-Färbung. I. Untersuchung der Farbstoffe. Acta Histochem 42:351–355Google Scholar
  16. Esterbauer H (1973) Beitrag zum quantitativen histochemischen Nachweis von Sulfhydrylgruppen mit der DDD-Färbung. II. Bestimmung von SH-Gruppen in unlöslichen Proteinen. Acta Histochem 47:94–105Google Scholar
  17. Esterbauer H, Nöhammer G, Schauenstein E, Weber P (1973) Beitrag zum quantitativen histochemischen Nachweis von Sulfhydrylgruppen mit der DDD-Färbung. III. Quantitative cytospektrometrische Bestimmungen an Ehrlich-Ascites-Tumorzellen. Acta Histochem 47:106–114Google Scholar
  18. Federici G, Duprè S, Matarese RM, Solinas SP, Cavallini D (1977) Is the alkaline cleavage of disulfide bonds in peptides an α-β elimination reaction or a hydrolysis?. Int J Peptide Protein Res 10:185–189Google Scholar
  19. Federici G, DiCola D, Sacchetta P, Dillio C, Del Boccio G, Polidoro G (1978) Reversible inactivation of tyrosine aminotransferase from guinea pig liver by thiol and disulfide compounds. Biochem Biophys Res Commun 81:650–655Google Scholar
  20. Friedman M (1973) The chemistry and Biochemistry of the sulfhydryl group in amino acids, peptides and proteins. Pergamon Press, OxfordGoogle Scholar
  21. Hofer E (1975) Wirkung SH-modifizierender Reagentien auf den synchronen Mitosezyklus von Physarum polycephalum. Doctors thesis, University Graz, AustriaGoogle Scholar
  22. Hupe DJ, Wu D (1980) Effect of charged substituents on rates of the thiol-disulfide interchange reaction. J Org Chem 45:3100–3103Google Scholar
  23. Isaacs J, Binkley F (1977) Glutathione dependent control of protein disulfide — sulfhydryl content by subcellular fractions of hepatic tissue. Biochim Biophys Acta 497:192–204Google Scholar
  24. Jensen EV (1959) Sulfhydryl-disulfide interchange. Science 130:1319–1323Google Scholar
  25. Jocelyn PC (1972) Biochemistry of the SH group. Academic Press, LondonGoogle Scholar
  26. Maddy AH (1961) 1-fluoro-2:4-dinitrobenzene as a cytochemical reagent. In: Danielli JF (ed) General cytochemical methods, vol 2. Academic Press, New YorkGoogle Scholar
  27. Mannervik B, Axelsson K (1980) Role of cytoplasmic thioltransferase in cellular regulation by thiol-disulfide interchange. Biochem J 190:125–130Google Scholar
  28. Modig H (1968) Cellular mixed disulphides between thiols and proteins, and their possible implication for radiation protection. Biochem Pharmacol 17:177–186Google Scholar
  29. Modig H, Révész L (1968) Cellular sulphydryl levels and the effect of two mercaptopyridoxine derivatives with differential radioprotective action. Arzneimittelforsch 18:1156–1157Google Scholar
  30. Nöhammer G, Schauenstein E, Weber P (1973) Quantification of selective bindung of 4-hydroxypentenal to structural protein thiols in Ehrlich tumor cells. An illustration of the efficiency of cytospectrometric evaluation of cytochemical results. J Histochem Cytochem 21:1082–1089Google Scholar
  31. Nöhammer G (1977) Quantitative cytospectrometrische Bestimmung von Proteinsulfhydrylgruppen und von reaktiven Disulfidgruppen mit der DDD-Echtblau B-Färbung an Ehrlich-Ascites-Tumorzellen. Acta Histochem 59:317–328Google Scholar
  32. Nöhammer G (1978) Die cytospectrometrische Proteinbestimmung mit der Tetrazonium-Kupplungsreaktion. I. Optimierung der Färbemethode. Acta Histochem 61:317–329Google Scholar
  33. Nöhammer G, Desoye G (1981) The cytospectrophotometrical determination of proteins by the tetrazonium coupling reaction. II. Calibration of the staining method. Acta Histochem 68:103–110Google Scholar
  34. Nöhammer G, Desoye G, Koschsorur G (1981) Quantitative cytospectrophotometrical determination of the total protein thiols with “Mercurochrom”. Optimization and calibration of the histochemical reaction. Histochemistry 71:291–300Google Scholar
  35. Olinescu R, Uleu C (1974) Studies on the SH-SS equilibrium in biological media. V. On the determination of sulfhydryl groups in tissue. Rev Roumaine Biochim 11:283–289Google Scholar
  36. Pearse AGE (1968) Histochemistry theoretical and applied, vol 1. Churchill, LondonGoogle Scholar
  37. Rindler R, Schauenstein E (1970) Über den Einfluß von 4-Hydroxypentenal auf den Gehalt proteingebundener und nicht-proteingebundener SH-Gruppen in Normal- und Tumorzellen. Z Naturforsch 25:739–744Google Scholar
  38. Robyt JF, Ackerman RJ, Chittenden CG (1971) Reaction of protein disulfide groups with Ellman's reagent: A case study of the number of sulfhydryl and disulfide groups in Aspergillus oryzae α-amylase, papain and lysozyme. Arch Biochem Biophys 147:262–269Google Scholar
  39. Ryle AP, Sanger F (1955) Disulfide interchange reactions. Biochem J 60:535–540Google Scholar
  40. Sillevis Smitt PAE, James J, Wisse JH (1968) The DDD-method in the cytophotometric quantitative estimation of protein-bound sulfhydryl groups in palatal smears. Acta Histochem 33:53–58Google Scholar
  41. Steven FS, Podrazky V (1978) Evidence for the inhibition of trypsin by thiols. The mechanism of enzyme-inhibitor complex formation. Eur J Biochem 83:155–161Google Scholar
  42. Torchinskii YuM (1974) Sulfhydryl and disulfide groups of proteins. Consultants bureau, New YorkGoogle Scholar
  43. Torchinskii YuM (1981) Sulfur in proteins. In: Metzler D (ed). Pergamon Press, OxfordGoogle Scholar
  44. Weber P (1974) Cytospektrometrische Bestimmung der Protein-SH-Gruppen in Tumor- und Normalzellen. Doctors Thesis, University Graz. AustriaGoogle Scholar

Copyright information

© Springer-Verlag 1982

Authors and Affiliations

  • G. Nöhammer
    • 1
  1. 1.Institute of BiochemistryUniversity of GrazGrazAustria

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