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.     

Ellman's reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination.

Accurate determination of thiol groups by means of Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid), DTNB] has been limited by uncertainty about the molar absorption coefficient of the dianion of the product, 2-nitro-5-thiobenzoic acid (TNB). A procedure is described for the purification of TNB by reduction of commercial DTNB followed by gel chromatography and crystallization. Pure DTNB is prepared by reoxidation of purified TNB followed by gel chromatography and crystallization. The molar absorption coefficient of the dianion of TNB is 14,150 at 412 nm in dilute aqueous salt solutions. This value was confirmed independently by reduction of purified DTNB with cysteine. Titration of sulphydryl groups with DTNB can be done at pH 7.27 where the thiol group of TNB is 99.8% in the intensely-colored conjugate base form while the hydroxide-promoted hydrolysis of DTNB is minimal.     

Reaction of ribosomal sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid)

The number of sulfhydryl groups in the Escherichia coli ribosome has been measured by titration with 5,5′-dithiobis(2-nitrobenzoic acid). Under denaturing conditions, there are 38.8 ± 1.0 titratable thiols per 70 S ribosome and 22.8 ± 0.3 and 12.9 ± 0.3 titratable thiols per 50 S and 30 S subunits, respectively. Three categories of thiol groups can be distinguished in the native 70 S ribosome, a “fast reacting” class of about 3 residues, a “slow reacting” class of about 10 residues and a “buried” class including about 26 residues. The addition of polyuridylic acid to reaction mixtures protects a fast-reacting thiol in the 30 S subunit belonging to protein S1.The addition of urea to ribosome solutions makes the buried residues titratable. Denaturation occurs as a sharp transition at a urea concentration between 4 and 4.5 m. Urea does not fully dissociate the ribosome into RNA and protein. Instead, in the case of the 30 S subunit, a slowly sedimenting particle forms in the presence of urea, containing roughly 65% of the normal amount of protein.     

Inter- and intramolecular crosslinks in histone H3 induced by 5,5'-dithiobis(2-nitrobenzoic acid).

The primary reaction between the cysteine residues of histone H3 and dithiobis-(nitrobenzoic acid) may be succeeded by thiol-disulfide interchange steps leading to intraor intermolecularly crosslinked H3 molecules. A chromatographic assay is applied to detect consecutive reactions of this type and is used to derive conditions by which they are promoted or suppressed. Elimination of the secondary interchange is shown to be essential if the kinetics of the reaction is to be interpreted.     

Electrostatic and conformational effects on the reaction of thiol groups of calf thymus histone H3 with 5,5'-dithiobis(2-nitrobenzoic acid).

The presence of highly basic proteins (histones or protamines), causes an increase in the rate of the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs₂) with the tripeptide model glutathione. This effect is explained by considering that polycationic molecules, such as histones or protamines, can attract the negatively charged reacting molecules, thus producing a catalytic effect. This effect disappears at high ionic strength due to a shielding of the charges; Urea causes a shift to the K_2(app)vs. pH curve for the histone H3-Nbs₂ reaction. This shift (2.1 units of pH for 8 m urea) indicates that urea denatures, at least to some extent, the tertiary structure of the microenvironments containing cysteine of histone H3, but it is unable to eliminate an unspecific electrostatic effect (similar to that caused by polycations in the GSH-Nbs₂ reaction), which also contributes to the increase of the reaction rate. Combined effects of urea and ionic strength on the reaction of GSH and of histone H3 with Nbs₂ gives rise to shifts of both curves of K_2(app)us. pH, approaching one to the other very closely. This is interpreted as due to the appearance of shielding effects on the electrostatic charges of the histone, and also of the small molecules. The greater efficiency of guanidine hydrochloride, compared to that of urea, in causing a shift of the rate constant curve of histone H3 is interpreted as due to a combined effect of denaturation and electrostatic shielding in the case of guanidine hydrochloride.     

Assay of glutathione reductase in crude tissue homogenates using 5,5'-dithiobis(2-nitrobenzoic acid)

A method for assaying glutathione reductase (GSH; EC 1.6.4.2) in crude plant extracts is described. The method is based on the increase in absorbance at 412 nm when 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is reduced by GSH. The effects of the following parameters on the assay were tested: various buffers, pH, buffer concentration, compounds commonly present in enzyme preparations, thiols, and the presence of another NADPH-dependent enzyme. The assay is more sensitive and less subject to interference than the widely used assay where NADPH oxidation is monitored. In particular, the specificity of DTNB allows assay of glutathione reductase in the presence of other NADPH-dependent enzymes and common protein extract contaminants.     

Glyoxalase II in Saccharomyces cerevisiae: in situ kinetics using the 5,5'-dithiobis(2-nitrobenzoic acid) assay.

The determination of glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) activity is usually accomplished by monitoring the decrease of absorbance at 240 nm due to the hydrolysis of S-d-lactoylglutathione. However, it was not possible, using this assay, to detect any enzyme activity in situ, in Saccharomyces cerevisiae permeabilized cells. Glyoxalase II activity was then determined by following the formation of GSH at 412 nm using 5,5'-dithiobis(2-nitrobenzoic acid). Using this method we characterized the kinetics of glyoxalase II in situ using S-d-lactoylglutathione as substrate and compared the results with those obtained for cell-free extracts. The specific activity was found to be (4.08 +/- 0.12) x 10(-2) micromol min-1 mg-1 in permeabilized cells and (3.90 +/- 0.04) x 10(-2) micromol min1 mg-1 in cell-free extracts. Kinetic parameters were Km 0.36 +/- 0.09 mM and V (7.65 +/- 0.59) x 10(-4) mM min-1 for permeabilized cells and Km 0.15 +/- 0.10 mM and V (7.23 +/- 1.04) x 10(-4) mM min-1 for cell-free extracts. d-Lactate concentration was also determined and increased in a linear way with permeabilized cell concentration. gamma-Glutamyl transferase (EC 2.3.2.2), which also accepts S-d-lactoylglutathione as substrate and hence could interfere with glyoxalase II assays, was found to be absent in Saccharomyces cerevisiae permeabilized cells.     

Use of a microplate reader in an assay of glutathione reductase using 5,5'-dithiobis(2-nitrobenzoic acid)

The use of 96-well microtiter plates and a programmable microplate reader to measure glutathione reductase in an assay based on reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by GSH generated from an excess of GSSG is described. Samples are prepared in 96-well plates and absorbance at 415 nm with a reference wavelength of 595 is determined every 30 s for 3 min. The rate of increase in absorbance is directly proportional to the amount of glutathione reductase in the sample. Activity in an unknown sample is determined from a standard curve. The assay is rapid and allows many small samples to be analyzed in replicates of two or more at the same time.     

The modification of sulfhydryl groups of glutamine synthetase from Bacillus stearothermophilus with 5, 5'-dithiobis(2-nitrobenzoic acid).

The SH groups of glutamine synthetase [EC 6.3.1.2] from Bacillus stearothermophilus were modified with 5, 5'-dithiobis(2-nitrobenzoic acid) in order to determine the number of SH groups in the molecule as well as the effect of the modification on the enzyme activity. Three SH groups per subunit were detected after complete denaturation of the enzyme with 6 M urea, one of which was essential for the enzyme activity in view of its reactivity with 5, 5'-dithiobis(2-nitrobenzoic acid) on addition of MgCl2 with loss of the activity. The CD spectra of the modified enzyme in the near ultraviolet region changed from that of the native enzyme, indicating that aromatic amino acid residues were affected by modification of the SH group. The fluorescence derived from tryptophanyl residue(s) was quenched depending on the extent of modification of the SH group, suggesting that the tryptophanyl residue(s) was located in the proximity of the SH group. The thermostability of the enzyme was remarkably decreased by modification of the SH group.     

Crosslinking by thiol disulfide interchange of 5,5'-dithiobis(2-nitrobenzoic acid)-treated light chain and heavy chain of rabbit skeletal myosin

Interchain disulfide crosslinks between the heavy-chain fragment in heavy meromyosin and myosin light chain 2, generated by 5,5'-dithiobis(2-nitrobenzoic acid (Nbs2), are formed under appropriate ionic conditions at neutral pH as revealed by liberation of the chromogenic 2-nitro-5-thiobenzoic acid. The presence of the original or of a slightly digested light chain 2 reduces the rate of the reaction of heavy meromyosin with Nbs2-modified light chain 2 by 32 - 39%, if Ca2+ is present. Dodecyl sulfate/polyacrylamide gel electrophoresis in absence of reducing agents shows that Nbs2-modified light chain 2 attaches to the heavy chain in the region of the 21-kDa fragment of heavy meromyosin, which contains the essential thiol groups and which has been located at the subfragment 1/subfragment 2 junction of myosin [Balint, M., Wolf, I., Tarcsafalvi, A., Gergely, J. and Sreter, F. A. (1978) Arch. Biochem. Biophys. 190, 793-799]. Modification of thiol-1 groups with iodoacetamide as well as crosslinking the thiol-1 and thiol-2 groups by the bifunctional reagent p-N,N'-phenylenedimaleimide prior to incubation with Nbs2-modified light chain 2 has no substantial effect on the crosslinking reaction. This indicates that other thiol groups are involved in the binding of Nbs2-modified light chain 2 to the heavy chain. An examination of K+, Ca2+, Mg2+ and actin-activated Mg2+ ATPase activities of heavy meromyosin that had been crosslinked with Nbs2-modified light chain 2 shows only a slight change in comparison with intact heavy meromyosin, indicating that crosslinking had not altered significantly the hydrolytic site. Crosslinking of Nbs2-modified light chain 2 to light-chain-2-deficient heavy meromyosin restored the original light-chain-2-dependent Ca2+ sensitivity of the tryptic fragmentation of heavy meromyosin, suggesting that crosslinking takes place at the proper binding site for light 2.     

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nanosecond time-resolved fluorescence kinetic studies of the 5,5'-dithiobis(2-nitrobenzoic acid) reaction with enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system

Enzyme I of the bacterial phosphotransferase system is a protein component which undergoes a temperature-dependent monomer/dimer equilibrium. Reaction of sulfhydryl residues with SH-specific reagents inhibits both activity and dimerization. There are four cysteine residues available in each subunit, one of which (Cys 502) is proximate to one of the two tryptophan residues (Trp 498). Previous studies revealed two major lifetimes and spectra, suggesting distinct environments for tryptophan. In this paper, we examine the dynamic quenching of tryptophanyl fluorescence that occurs when an energy transfer acceptor, thio-2-nitrobenzoic acid (TNB), is covalently attached to the sulfhydryl groups. More precisely, we have traced the recovery of nativelike fluorescence lifetime components (and the concomitant loss of "reduced lifetime" amplitudes) that accompanies TNB release. The course of lifetime changes seen when a reducing reagent removes the quencher may be sensitive to a variety of effects, including different SH affinities, different proximities to Trp, changing availability for dimerization, or conformational changes. The prospective value of separating each lifetime component from the mixture is illustrated.