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

Abstract

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 specificity 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 Nöhammer (1978) and calibrated by Nöhammer et al. (1981), was used.

Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides

“…As discussed above, the incubation of fixed cells in ethanolic buffer pH 8.6 (without DDD; controls) leads to a loss of protein mixed disulfides caused by the disulfide exchange reaction (Nöhammer 1982;Rossi et al 1995). The result is the formation of new protein disulfides paralleled by an equimolar artificial loss of protein thiols (Fig.…”

“…The DDD method DDD reacts in a time-dependent manner with protein thiols and with reactive disulfides (Nöhammer 1982). During the first 7 h DDD reacts preferentially with protein thiols, generating protein mixed disulfides.…”

“…To answer the question whether 1 or 2 mol of mercurochrom are bound per mol disulfide, the DDD reaction was used (Nöhammer 1982). DDD reacts with protein thiols in a disulfide exchange reaction and, at a much slower rate, with reactive mixed disulfides in a disulfide interchange reaction.…”

“…The thiol-disulfide exchange reaction for the removal of protein mixed disulfides (mainly PSSG) as described by Nöhammer (1982) for fixed cell preparations and by Rossi et al (1995) for protein solutions according to: PSSG+PSH=PSSP+GSH was performed by incubation of freshly fixed preparations of EATC in 0.1 M TRIS buffer pH 7.4 for 1 h followed by both Merc and MCN staining.…”

“…For controls, DDD solution was replaced by ethanolic buffer pH 8.6. After the last washing step the preparations were stained using either the Merc and MCN staining methods, or the FbB-azo coupling step of the DDD-FbB staining method (Nöhammer 1982).…”