Characterization of a new sulfhydryl group reagent: 6,6′-Diselenobis-(3-Nitrobenzoic acid), a selenium analog of Ellman's reagent

A new reagent, 6,6′-diselenobis-(3-nitrobenzoic acid) (DSNB) has been synthesized and is shown to be useful for quantitative estimation of sulfhydryl groups in proteins. This reagent, which is a selenium analog of Ellman's reagent, reacts specifically and quantitatively with thiol groups of proteins to yield a selenenyl sulfide and the dianion of 3-nitro-6-selenobenzoic acid. The molar absorption coefficient of the chromophoric dianion is 10,000 at 432 nm in dilute aqueous solutions. The titration can best be performed at pH 8.20 where >98% of 3-nitro-6-selenobenzoic acid is in the form of the intensely colored dianion. Sulfhydryl content determinations by this reagent of reduced and denatured ribonuclease, reduced and denatured lysozyme, native papain, and native and denatured thymidylate synthetase are compared with those from corresponding 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) titrations. The reagent was found to inactivate thymidylate synthetase, an enzyme with essential sulfhydryl groups. Unlike DTNB which undergoes alkaline decomposition of pH values greater than 9, DSNB was found to be stable to hydrolysis, even in 0.05 m NaOH.     

Molar absorption coefficients for the reduced Ellman reagent: reassessment

The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.     

Disulfide formation within the regulatory light chain of skeletal muscle myosin

Thiol-disulfide exchange reactions between myosin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) lead to the formation of 5-thio-2-nitrobenzoic acid (TNB)-mixed disulfides as well as to protein disulfide bonds. After incubation with DTNB, myosin was treated with an excess of N-ethylmaleimide (NEM) before electrophoretic analysis of the protein subunits in sodium dodecyl sulfate (SDS) without prior reduction by dithiothreitol (DTT). Without NEM treatment, thiol-disulfide rearrangement reactions occurred in the presence of SDS between the residual free thiols and DTNB. In the absence of divalent metal ions at 25 degrees C, DTNB was shown to induce an intrachain disulfide bond between Cys-127 and Cys-156 of the RLC. This intrachain cross-link restricts partially the unfolding of the RLC in SDS and can be followed as a faster migrating species, RLC'. Densitometric evaluation of the electrophoretic gel patterns indicated that the stoichiometric relation of the light chains (including RLC and RLC') remained unchanged. The two cysteine residues of the fast migrating RLC' were no more available for reaction with [14C]NEM, but upon reduction with DTT, the electrophoretic mobility of the RLC' reverted to that of unmodified RLC and of the RLC modified with two TNB groups. Ca2+ or Mg2+ was able to prevent this disulfide formation in the RLC of myosin by 50% at a free ion concentration of 1.1 X 10(-8) and 4.0 X 10(-7) M, respectively, at 25 degrees C and pH 7.6. Intrachain disulfide formation of RLC never occurred in myosin at 0 degree C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Essential sulfhydryl groups of rat liver monoamine oxidase.

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 degrees C are 5.80 times 10(-4) M and 4.35 times 10(-5) M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO. The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 +/- 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 to total sulfhydryl groups (n = 2) on denaturation with 8 M urea.     

The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5'-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14--16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.     

Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes

The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306M(-1)min(-1)) than the oximate ion of HI-6 (22M(-1)min(-1)). However, the rate constants obtained with the Ellman method were 84M(-1)min(-1) for K027 and 22M(-1)min(-1) for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.     

The reactivity of the thiol groups of calf thymus deoxyribonucleohistone

The reactivities of the two cysteine thiol groups of calf thymus F3 histone were investigated using 5,5'-dithiobis-[2- nitrobenzoic acid], (DTNB). In isolated histone, both thiol groups were available for reaction. However, analysis of reaction profiles of native deoxyribonucleohistone, (DNH), in various solvent conditions, together with gel electrophoresis studies of DNH modified with DTNB, showed that only one of the thiol groups is normally modified by the reagent. If NaCl is present (above 1.OM) the other thiol group can also be modified. The reactivities of both groups were largely independent of the degree of DNH supercoiling and of the binding of F3 to the DNA.     

The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5′-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14–16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.     

Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis

Deoxycytidylate (dCMP) hydroxymethylase from Escherichia coli infected with a T-4 bacteriophage amber mutant has been purified to homogeneity. It is a dimer with a subunit molecular weight of 28,000. Chemical modification of the homogeneous enzyme with N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to complete loss of enzyme activity. dCMP can protect the enzyme against NEM inactivation, but the dihydrofolate analogues methotrexate and aminopterin alone do not afford similar protection. Compared to dCMP alone, dCMP plus either methotrexate or aminopterin greatly enhances protection against NEM inactivation. DTNB inactivation is reversed by dithiothreitol. For both reagents, inactivation kinetics obey second-order kinetics. NEM inactivation is pH dependent with a pKa for a required thiol group of 9.15 +/- 0.11. Complete enzyme inactivation by both reagents involves the modification of one thiol group per mole of dimeric enzyme. There are two thiol groups in the totally denatured enzyme modified by either NEM or DTNB. Kinetic analysis of NEM inactivation cannot distinguish between these two groups; however, with DTNB kinetic analysis of 2-nitro-5-thiobenzoate release shows that enzyme inactivation is due to the modification of one fast-reacting thiol followed by the modification of a second group that reacts about 5-6-fold more slowly. In the presence of methotrexate, the stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group. It appears that the two equally sized subunits are arranged asymmetrically, resulting in one thiol-containing active site per mole of dimeric enzyme.     

The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine.

Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.     

Kinetics of modification of the mitochondrial succinate-ubiquinone reductase by 5,5′-dithiobis-(2-nitro-benzoic acid)

The kinetic theory of the substrate reaction during modification of enzyme activity previously described by Tsou [Tsou (1988),Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381–436] has been applied to a study of the kinetics of the course of inactivation of the mitochondrial succinate-ubiquinone reductase by 5,5′-dithiobis-(2-nitro-benzoic acid) (DTNB). The results show that the inactivation of this enzyme by DTNB is a conformation-change-type inhibition which involves a conformational change of the enzyme before inactivation. The microscopic rate constants were determined for the reaction of the inactivator with the enzyme. The presence of the substrate provides marked protection of this enzyme against inactivation by DTNB. The modification reaction of the enzyme using DTNB was shown to follow a triphasic course by following the absorption at 412 nm. Among these reactive thiol groups, the fast-reaction thiol group is essential for the enzyme activity. The results suggest that the essential thiol group is situated at the succinate-binding site of the mitochondrial succinate-ubiquinone reductase.     

Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. II. Purification and characterization of b-type cytochrome and NADH-ferricyanide reductase from Ascaris muscle microsomes.

A b-type cytochrome and NADH-ferricyanide (FC) reductase were solubilized from Ascaris muscle microsomes by detergents and purified by column chromatography. The purified b-type cytochrome displayed absorption bands at 560 (alpha-peak), 525 (beta-peak), and 424 nm (gamma-peak), with a marked shoulder at 555 nm in the reduced from, 415 nm (gamma-peak) in the oxidized form. This absorption spectrum was different from that of rat liver microsomal cytochrome b5. The molecular weight was estimated to be about 100,000 by SDS-polyacrylamide gel electrophoresis, and the absorption spectrum of alkaline pyridine ferrohemochrome suggested that the prosthetic group of this cytochrome is protoheme. The molecular weight of the purified NADH-FC reductase was estimated to be about 55,000 by SDS-polyacrylamide gel electrophoresis. The purified reductase required NADH as a specific electron donor. The reductase efficiently reduced some redox dyes with NADH, but the reduction of cytochrome c was much slower. The purified reductase, like the membrane-bound reductase, was not inhibited by thiol reagents.     

5,5′-Dithiobis-(2-Nitrobenzoic Acid) as a Probe for a Non-Essential Cysteine Residue at the Medium Chain Acyl-Coenzyme a Dehydrogenase Binding Site of the Human ‘Electron Transferring Flavoprotein’ (ETF)

Abstract

Human ‘electron transferring flavoprotein’ (ETF) was inactivated by the thiol-specific reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.     

Euglena gracilis cadmium-binding protein-II contains sulfide ion

Sulfide ions are a constituent of the cadmium-binding protein-II in the alga Euglena gracilis. Their presence was demonstrated by the methylene blue assay, by acid labilization induced reductions in the Cd-S charge transfer band at 254 nm and by reactions with the thiol reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). Direct reduction of DTNB by sulfide and precipitation of CdS yield a complex stoichiometry for the DTNB reaction. The S2-/Cd2+ ratios determined, 1.25 +/- 0.10 (methylene blue) and 1.37 +/- 0.16 (DTNB), are in good agreement.     

Purification and characterization of recombinant human interleukin-2 produced in Escherichia coli

Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli was purified to apparent homogeneity by cation exchange chromatography and reverse phase high performance liquid chromatography. The amino acid composition, amino terminal amino acid sequence, and carboxyl terminal amino acid were consistent with those deduced from the cDNA sequence. Besides the molecular species with the amino terminal Ala, the purified preparation contained another species having an additional Met residue at the amino terminus corresponding to the initiation codon AUG. The molar absorption coefficient of rIL-2 was determined to be 9.58 X 10(3) M-1 cm-1 at 280nm in water. Ultracentrifugal analyses revealed that it existed as a monomeric form in 0.1 M NaCl. The apparent sedimentation coefficient (S20,w) was calculated to be 1.8 S.     

Abolition of ATPase activities of skeletal myosin subfragment 1 by a new selective proteolytic cleavage within the 50-kilodalton heavy chain segment

We have isolated and chemically characterized several 5-thio-2-nitrobenzoate-subfragment 1 derivatives (TNB-S-1) generated by the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB, up to 10-fold molar excess) with native S-1, N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-S-1 (AEDANS-S-1), and N,N'-p-phenylenedimaleimide-S-1 (pPDM-S-1) at 4 degrees C, pH 8.0. The reaction of the reagent with AEDANS-S-1, which has a blocked -SH1 group, induced the formation of an intramolecular cystine disulfide between two vicinal -SH groups in S-1; in contrast, the treatment of pPDM-S-1 with DTNB resulted in the formation of TNB mixed disulfides only. The incorporation of the TNB groups (up to 3 mol/mol of S-1) into the native or premodified S-1 led to a local conformational change in the 50K heavy chain region that was fully reversed upon disulfide reduction. Exploiting this peculiarity of the DTNB-modified S-1's, we have realized a highly selective proteolysis of the S-1 heavy chain by thrombin and chymotrypsin, which do not act at all on the normal S-1. The 95K heavy chain was cut by thrombin into two fragments with apparent masses of 68K and 30K, whereas the "connector segments" and the light chains were unaffected. The two new fragments were issued from a primary peptide-bound cleavage between Lys-560 and Ser-561 within the amino acid sequence of the 50K region (M. Elzinga, personal communication).(ABSTRACT TRUNCATED AT 250 WORDS)     

DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography.

A new method of affinity chromatography using blue dextran-Sepharose 4B resin was established to purify NADP+-dependent isocitrate dehydrogenase [EC 1.1.1.42] from Bacillus stearothermophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidine-HCl the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the alpha-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluorescence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residue(s). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mg2+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heating at 65 degrees was observed in the presence of substrate and Mg2+, but the thermostability of the enzyme was markedly reduced by modification of the SH groups.     

Conformation and stability of thiol-modified bovine beta-lactoglobulin

Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. Beta-lactoglobulin A has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCI at pH 7.5, producing a modified beta-lactoglobulin (TNB-bIg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-bIg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-bIg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the alpha-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-bIg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-bIg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-bIg were monomeric. The unfolding transition of TNB-bIg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-bIg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-bIg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-bIg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.     

Quantitative determination of sulfhydryl groups with "mercurochrome" (author's transl)

Dibrommercuryfluoresceine (DBMF) reacts stoichiometrically and quantitatively with the thiol group of cysteine, glutathione and thioglycolic acid respectively, at pH 7.0. Polarographical and spectrometrical titrations clearly show that in the spectra of the investigated mercaptides the wave length of the first absorption maximum of DMBF (507 nm) remains unchanged but the molar extinction coefficient increases by approximately 20%. Serum albumin, ovalbumin, beta-lactoglobulin and glyceraldehydephosphatedihydrogenase, after incubation with DBMF, form adducts with the dye from which the pure mercaptide complexes were separated by means of column chromatogrphy. These complexes were separated by means of column chromatography. These complexes show a bathochromic shift (520 nm) of the dye band which is decreased now by 50%. The molar extinction coefficient epsilon 520 has been determined from 32,000 to 33,850. On the basis of these values SH-contents of the four proteins were obtained which are in good accordance with data previously published in the literature. No selective reaction, f.i. with more accessible or/and reactive SH-groups was observed. After 30 min incubation with DBMF and washing with isotonic phosphate buffer, native animal tumor cells show in the main absorption band the bathochromically shifted dye maximum. A first temptative estimation of the protein SH-groups yielded 1.7-2.1 X 10(-14) mole SH/single cell. This result lies between the SH-content determined microspectrometrically on cells stained with DDD-Fast Blue B (1.1-1.55 X 10(-14)) and macroscopically on cell homogenates with DTNB (3.1 X 10(-14)). Up to now, no certain information can be given whether or to what extent unspecific absorption effects possibly might be involved in the data obtained with DBMF treated cells, but interaction with nucleic acids can be excluded with certainty on the basis of relevant model experiments.