Quantitative EPR Spectroscopy Research Articles

Valence Tautomerism in Chromium Half-Sandwich Triarylmethylium Dyads

Valence tautomerism (VT) may occur if a molecule contains two chemically different redox-active units, which differ only slightly in their intrinsic redox potential. Herein, we present three new half-sandwich complexes [(η6-arene)Cr(CO)2L]+ with a triarylmethylium substituent appended to the π-coordinated arene and different coligands L (L = CO, P(OPh)3, PPh3, 1+–3+) at the chromium atom. Ligand substitution purposefully lowers the half-wave potential for chromium oxidation and thereby the redox potential difference towards tritylium reduction. For the PPh3-substituted complex 3+, cyclic voltammetry measurements indicate that chromium oxidation and tritylium reduction occur at (almost) the same potential. This renders the diamagnetic Cr(0)-C6H4-CAr2+ form 3+, and its paramagnetic diradical Cr(I)+•-C6H4-CAr2• valence tautomer 3+•• energetically nearly degenerate. Temperature-dependent IR spectroscopy indeed shows two pairs of carbonyl bands that are assignable to a Cr(0) and a Cr(I) species, coexisting in a T-dependent equilibrium with almost equal quantities for both at −70 °C. The diradical form with one unpaired spin at the trityl unit engages in a monomer ⇌ dimer equilibrium, which was investigated by means of quantitative EPR spectroscopy. The diradical species 1+••–3+•• were found to be highly reactive, leading to several identified reaction products, which presumably result from hydrogen atom abstraction via the trityl C atom, e.g., from the solvent.

Tailoring Valence Tautomerism by Using Redox Potentials: Studies on Ferrocene-Based Triarylmethylium Dyes with Electron-Poor Fluorenylium and Thioxanthylium Acceptors.

Three new electrochromic ferrocenyl triarylmethylium dyes with fluorenylium (1 a +, 1 b +) or thioxanthylium (1 c +) residues were selected in order to keep the intrinsic differences of redox potentials for ferrocene oxidation and triarylmethylium reduction small and to trigger valence tautomerism (VT). UV/Vis/NIR and quantitative EPR spectroscopy identified paramagnetic diradical isomers 1 a ..+–1 c ..+ alongside diamagnetic forms 1 a +–1 c +, which renders these complexes magnetochemical switches. The diradical forms 1 a ..+–1 c ..+ as well as the one‐electron‐reduced triarylmethyl forms of the complexes were found to dimerize in solution. For radical 1 a ., dimerization occurs on the timescale of cyclic voltammetry; this allowed us to determine the kinetics and equilibrium constant for this process by digital simulation. Mößbauer spectroscopy indicated that 1 a + and 1 b + retain VT even in the solid state. UV/Vis/NIR spectro‐electrochemistry revealed the poly‐electrochromic behaviour of these complexes by establishing the distinctly different electronic absorption profiles of the corresponding oxidized and reduced forms.

Synthesis and Thin Films of Thermally Robust Quartet (S = 3/2) Ground State Triradical.

High-spin (S = 3/2) organic triradicals may offer enhanced properties with respect to several emerging technologies, but those synthesized to date typically exhibit small doublet quartet energy gaps and/or possess limited thermal stability and processability. We report a quartet ground state triradical 3, synthesized by a Pd(0)-catalyzed radical-radical cross-coupling reaction, which possesses two doublet-quartet energy gaps, ΔEDQ ≈ 0.2-0.3 kcal mol-1 and ΔEDQ2 ≈ 1.2-1.8 kcal mol-1. The triradical has a 70+% population of the quartet ground state at room temperature and good thermal stability with onset of decomposition at >160 °C under an inert atmosphere. Magnetic properties of 3 are characterized by SQUID magnetometry in polystyrene glass and by quantitative EPR spectroscopy. Triradical 3 is evaporated under ultrahigh vacuum to form thin films of intact triradicals on silicon substrate, as confirmed by high-resolution X-ray photoelectron spectroscopy. AFM and SEM images of the ∼1 nm thick films indicate that the triradical molecules form islands on the substrate. The films are stable under ultrahigh vacuum for at least 17 h but show onset of decomposition after 4 h at ambient conditions. The drop-cast films are less prone to degradation in air and have a longer lifetime.

Diagnosis of Nitrosative Stress by Quantitative EPR-Spectroscopy of Epidermal Cells

Quantitative assay of nitric oxide (NO) and iron (Fe) was carried out by the electron paramagnetic resonance (EPR) method in epidermal derivative (hair) of 45 liquidators of the Chernobyl accident, 30 employees of radiological institutions having contact with radiation, and 50 students of Vladimir State University, most of which were diagnosed with iron deficiency, namely iron deficiency anemia (4 persons) and latent iron deficiency (34 persons). Measurements were performed on a Radiopan EPR-spectrometer. It was found that the method of quantitative EPR-spectroscopy using diethyldithiocarbamate as a trap in vitro could be successfully used to measure NO-radical activity in such a biological substrate as hair. It was found that the intensity of NO-radical signal in the spectrogram depends on the Fe level (not only in the analyzed substrate, but also in the whole organism).

Characterization of the Mechanism and Magnitude of Cytoglobin-mediated Nitrite Reduction and Nitric Oxide Generation under Anaerobic Conditions

Cytoglobin (Cygb) is a recently discovered cytoplasmic heme-binding globin. Although multiple hemeproteins have been reported to function as nitrite reductases in mammalian cells, it is unknown whether Cygb can also reduce nitrite to nitric oxide (NO). The mechanism, magnitude, and quantitative importance of Cygb-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, spectrophotometric measurements, and chemiluminescence NO analyzer studies were performed. Under anaerobic conditions, mixing nitrite with ferrous-Cygb triggered NO formation that was trapped and detected using EPR spin trapping. Spectrophotometric studies revealed that nitrite binding to ferrous-Cygb is followed by formation of ferric-Cygb and NO. The kinetics and magnitude of Cygb-mediated NO formation were characterized. It was observed that Cygb-mediated NO generation increased linearly with the increase of nitrite concentration under anaerobic conditions. This Cygb-mediated NO production greatly increased with acidosis and near-anoxia as occur in ischemic conditions. With the addition of nitrite, soluble guanylyl cyclase activation was significantly higher in normal smooth muscle cells compared with Cygb knocked down cells with Cygb accounting for ∼40% of the activation in control cells and ∼60% in cells subjected to hypoxia for 48 h. Overall, these studies show that Cygb-mediated nitrite reduction can play an important role in NO generation and soluble guanylyl cyclase activation under hypoxic conditions, with this process regulated by pH, oxygen tension, nitrite concentration, and the redox state of the cells.

Formation and Reactivity of Chromium(V)−Thiolato Complexes: A Model for the Intracellular Reactions of Carcinogenic Chromium(VI) with Biological Thiols

The nature of the long-lived EPR-active Cr(V) species observed in cells and biological fluids exposed to carcinogenic Cr(VI) has been definitively assigned from detailed kinetic and spectroscopic analyses of a model reaction of Cr(VI) with p-bromobenzenethiol (RSH) in the presence or absence of cyclic 1,2-diols (LH(2)) in aprotic or mixed solvents. The first definitive structures for Cr(V) complexes with a monodentate thiolato ligand, [Cr(V)O(SR)(4)](-) (g(iso) = 1.9960, A(iso) = 14.7 x 10(-4) cm(-1)), [Cr(V)OL(SR)(2)](-) (g(iso) = 1.9854, A(iso) = (15.8-16.2) x 10(-4) cm(-1)) and [Cr(V)(O)(2)(SR)(2)](-) (g(iso) = 1.9828, A(iso) = 6.8 x 10(-4) cm(-1)) were assigned by EPR spectroscopy and electrospray mass spectrometry. The unusually low A(iso) ((53)Cr) value for the latter species is consistent with its rare four-coordinate, bis-oxido structure. The [Cr(V)OL(SR)(2)](-) species are responsible for the transient g(iso) approximately 1.986 EPR signals observed in living cells and animals treated with Cr(VI) (where RSH and LH(2) are biological thiols and 1,2-diols, respectively). For the first time, concentrations of Cr(V) intermediates formed during the reduction of Cr(VI) were determined by quantitative EPR spectroscopy, and a detailed reaction mechanism was proposed on the basis of stochastic simulations of the kinetic curves for Cr(V) species. A key feature of the proposed mechanism is the regeneration of Cr(V) species in the presence of Cr(VI) through the formation of organic free radicals, followed by the rapid reactions of the formed radicals with Cr(VI). The concentration of Cr(V) grows rapidly at the beginning of the reaction, reaches a steady-state level, and then drops sharply once Cr(VI) is spent. Similar mechanisms are likely to operate during the reduction of Cr(VI) in biological environment rich in reactive C-H bonds, including the oxidative DNA damage by Cr(V) intermediates.

Radioprotective thermally generated free-radical dextrins

Effect of doses of the X-ray radiation from 0 to 400 Gy upon granular cornstarch and dextrins (British gums, BG) thermally generated from it at 230–300°C was recognized with quantitative EPR and IR absorption spectroscopy, molecular mass distribution in the depolymerization products, Scanning Electron Microscopy, and X-ray diffractometry. Fractal analysis of the profiles of molecular mass distribution showed that the depolymerization involved debranching of amylopectin. Roasting of cornstarch produced BG which differed in concentration and EPR parameters of stable free radicals from BG generated by X-ray radiation. Two types of stable free radicals, with Gaussian and Lorentzian shapes of EPR signals, were recognized. The shapes of the signals and temperature dependence on free radical intensity indicated exchanging interactions of the antiferromagnetic type, causing partial quenching of the spins at −196°C (77K). Upon X-ray irradiation, new radicals were generated, the number and stability of which strongly depended on the types of radicals present before irradiation. These radicals slowly ceased because of a repolymerization of BG on storage.

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded ∼1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed ∼1 mol of the S = 1/2 signal from the reduced [2Fe–2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K M- and apparent k cat-values of 0.15 μM and 3.0 s −1, respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.

Analysis of the longitudinal, “sloping plateau” effect of a planar sample in a single TE 102 rectangular electron paramagnetic resonance cavity

The response of the cavity to the movement of a concentrically positioned “over full-length cavity” planar sample of variable length, L = 30, 40, and 50 mm, and width, w = 4 , 6, and 8 mm, along the vertical, common sample–cavity x-axis in a Bruker single TE 102 rectangular cavity (longitudinal effect) has been analysed. The observed dependence of the EPR signal intensity, Ipp, on the sample position, x, showed the following: (i) A plateau, over which the Ipp value remained constant within experimental error of 0.62–2.48%, for sample length of 30 and 40 mm. (ii) An “sloping plateau” region, which could be approximated by a linear function (correlation, R = 0.83–0.93), for sample length of 50 mm. (iii) An additional oscillating component superimposed on the “sloping plateau” region. The trends of these dependencies with the plateau and “sloping plateau” region were independent of the planar sample width, w , and its angular position, ϕ, in the rectangular cavity. But, the EPR signal amplitude on the plateau and “sloping plateau” region obtained for the sample positioned at angles, ϕ = 0° and ϕ = 180° (which corresponded to the experimental configuration, in which the planar sample area is parallel to the waveguide in the microwave cavity) was ca 1.8 times higher compared to those obtained for the sample positioned at angles ϕ = 90° and ϕ = 270° (which corresponded to the experimental configuration, in which the planar sample area is perpendicular to the waveguide). Theoretical predictions of the experimental dependencies of the EPR signal intensity versus sample position along the common sample–cavity x-axis can be calculated using the “sine-squared” function (given by longitudinal integral) modified for an “over full-length cavity” planar sample, and the correlation between observed and theoretically computed dependencies is very good. Each of the above-mentioned phenomenon could be a serious source of significant errors in quantitative EPR spectroscopy. For maximum accuracy in quantitative EPR an “over full-length cavity” planar sample should be compared to a reference sample of identical length which must be identically positioned in the microwave cavity.

EPR Studies of Thermally Treated Oxidized Corn Starch

AbstractThe formation of radicals in oxidized corn starch at 20–250°C in air was investigated by quantitative EPR spectroscopy. It was found that the presence of carboxyl groups in the oxidized starch increases the starch's ability to generate radical species. Introducing Cu2+ ions into the starch, as a catalyst or as an EPR probe, leads to the formation of copper‐carboxyl complexes containing two carboxyl ligands. Drastic increase in the number of radical species at about 250°C is accompanied by a decrease in the intensity of EPR signal of Cu2+‐carboxyl complexes, indicating that the thermal degradation of oxidized corn starch occurs with the formation of radicals, probably via breaking the chemical bonds in the carboxyl groups.

Mechanistic implications for the formation of the Diiron cluster in ribonucleotide reductase from quantitative EPR spectroscopy

Mechanistic implications for the formation of the Diiron cluster in ribonucleotide reductase from quantitative EPR spectroscopy

Mechanistic implications for the formation of the diiron cluster in ribonucleotide reductase provided by quantitative EPR spectroscopy.

The small subunit of Escherichia coli ribonucleotide reductase (R2) is a homodimeric (betabeta) protein, in which each beta-peptide contains a diiron cluster composed of two inequivalent iron sites. R2 is capable of reductively activating O(2) to produce a stable tyrosine radical (Y122*), which is essential for production of deoxyribonucleotides on the larger R1 subunit. In this work, the paramagnetic Mn(II) ion is used as a spectroscopic probe to characterize the assembly of the R2 site with EPR spectroscopy. Upon titration of Mn(II) into samples of apoR2, we have been able to quantitatively follow three species (aquaMn(II), mononuclear Mn(II)R2, and dinuclear Mn(2)(II)R2) and fit each to a sequential two binding site model. As previously observed for Fe(II) binding within apoR2, one of the sites has a greater binding affinity relative to the other, K(1) = (5.5 +/- 1.1) x 10(5) M(-)(1) and K(2) = (3.9 +/- 0.6) x 10(4) M(-)(1), which are assigned to the B and A sites, respectively. In multiple titrations, only one dinuclear Mn(2)(II)R2 site was created per homodimer of R2, indicating that only one of the two beta-peptides of R2 is capable of binding Mn(II) following addition of Mn(II) to apoR2. Under anaerobic conditions, addition of only 2 equiv of Fe(II) to R2 (Fe(2)(II)R2) completely prevented the formation of any bound MnR2 species. Upon reaction of this sample with O(2) in the presence of Mn(II), both Y122* and Mn(2)(II)R2 were produced in equal amounts. Previous stopped-flow absorption spectroscopy studies have indicated that apoR2 undergoes a protein conformational change upon binding of metal (Tong et al. J. Am. Chem. Soc. 1996, 118, 2107-2108). On the basis of these observations, we propose a model for R2 metal incorporation that invokes an allosteric interaction between the two beta-peptides of R2. Upon binding the first equiv of metal to a beta-peptide (beta(I)), the aforementioned protein conformational change prevents metal binding in the adjacent beta-peptide (beta(II)) approximately 25 A away. Furthermore, we show that metal incorporation into beta(II) occurs only during the O(2) activation chemistry of the beta(I)-peptide. This is the first direct evidence of an allosteric interaction between the two beta-peptides of R2. Furthermore, this model can explain the generally observed low Fe occupancy of R2. We also demonstrate that metal uptake and this newly observed allosteric effect are buffer dependent. Higher levels of glycerol cause loss of the allosteric effect. Reductive cycling of samples in the presence of Mn(II) produced a novel mixed metal Fe(III)Mn(III)R2 species within the active site of R2. The magnitude of the exchange coupling (J) determined for both the Mn(2)(II)R2 and Fe(III)Mn(III)R2 species was determined to be -1.8 +/- 0.3 and -18 +/- 3 cm(-)(1), respectively. Quantitative spectral simulations for the Fe(III)Mn(III)R2 and mononuclear Mn(II)R2 species are provided. This work represents the first instance where both X- and Q-band simulations of perpendicular and parallel mode spectra were used to quantitatively predict the concentration of a protein bound mononuclear Mn(II) species.

Analysis of the Radial and Longitudinal Effect in a Double TE104 and a Single TE102 Rectangular Cavity

The response of the cavity to the rotation of a pointlike sample in the horizontal (y–z) plane passing through the center of the Bruker double TE104 and single TE102 rectangular cavities in concentric circles of radii ρ = 0, 1, 2, 3, 4, and 5 mm from the cavity center (radial effect) has been analyzed. The experimentally observed dependencies of the EPR signal intensity, Ipp, showed the following: (i) for ρ = 0 mm (a sample position in the cavity center), Ipp is independent of the angle of rotation; (ii) for ρ = 1, 2, and 3 mm, the Ipp dependence progressively changes from circular to oval; (iii) when the radius is further increased to ρ = 4 and 5 mm, the Ipp dependence changes dramatically, giving a figure eight shape. These experimental observations are in very good agreement with the theoretical calculations, in which the response is modeled using modified Cassinian curves, K(ρ, φ). Similar trends were observed for any position of the horizontal (y–z) plane at which the sample is situated along the vertical x axis of the cavity; however, the amplitude of the signal decreases with increase in the absolute value of the x coordinate, ‖x‖. The variation in the signal amplitude along the cavity x axis (longitudinal effect) can be calculated theoretically using a modified sine-squared curve, G(x). In general, the response of the cavity to a pointlike sample situated at any position, P(ρ, φ, x), can be represented as a product of the mentioned Cassinian curve, K(ρ, φ), and sine-squared curve, G(x), giving for the signal intensity Ipp(ρ, φ, x) ∝ K(ρ, φ)G(x). The response to a large cylindrical sample which is concentrically situated on the cavity x axis can then be obtained by integrating the above product, K(ρ, φ)G(x), over the sample volume. The nonlinear radial effect may give rise to a serious source of systematic error in quantitative EPR spectroscopy and shows that accurate and precise positioning of the sample in the microwave cavity is essential.

Quantitative EPR spectroscopy: Comparison between primary standards and application to MgO-MnO and α-Al2O3-Cr2O3 solid solutions

To study their reliability as primary standards in the quantitative EPR spectroscopy, a large series of pure paramagnetic compounds with known spin concentrations, whose spectra vary considerably in intensity, shape, structure and overall width are compared. The paramagnetic species examined as pure solid compounds and solutions, were free radicals (DPPH and TEMPO), vanadyl and Cu2+ ions (S = 1/2), Cr3+ (S = 3/2) and Mn2+ (5 = 5/2) ions. The quantitative EPR findings suggest that all theS = 1/2 paramagnetic compounds investigated and MnSO4 · H2O (S = 5/2) are reliable primary standards. By contrast, none of the pure Cr3+ compounds proved useful as primary standards because of their large fine-structure terms or high Neel temperature that invalidated the simple Curie law. Application of quantitative EPR in the study of dilute MgO-MnO and α-Al2O3-Cr2O3 solid solutions, focussing on the circumstances making paramagnetic species undetectable, is reported. In MgO-MnO solid solutions of high surface area, detection problems arising from the variation of local site symmetry can be circumvented and almost all Mn2+ are detected only by reducing the surface area. In concentrated α-Al2O3-Cr2O3 solid solutions, magnetic interactions lead to paramagnetic species being undetectable.

Analysis of the Movement of Line-like Samples of Variable Length along theX-Axis of a Double TE104and a Single TE102Rectangular Cavity

Movement of line-like samples with lengths from 5 to 50 mm along thex-axis of the double TE104rectangular cavity has been analyzed. The observed dependencies of the EPR signal intensity versus sample position showed: (i) a sharp maximum for sample lengths from 5 to 20 mm; (ii) a plateau, over which the EPR signal intensity remained constant within experimental errors of 0.26–1.07%, for lengths from 30 to 40 mm; and (iii) a “sloping plateau,” which could be approximated by the linear function (correlation,r= 0.98) for sample length 50 mm. Theoretical values of the experimentally observed dependencies of the intensity versus sample position were calculated using the modified sine-squared function and the correlation between observed and theoretically predicted dependencies is very good. The experimental dependence of the EPR signal intensity versus the sample length for samples situated at the same point in the cavity was nonlinear with a maximum for the 40-mm sample. The dependence of the EPR signal intensity upon the movement of a large cylindrical sample (o.d. 4 mm and length 100 mm) along thex-axis of the cavity was similar to that found for the 50-mm sample. However, an additional oscillating signal superimposed on the sloping plateau was observed. The presence of a large sample fixed in the complementary cavity of the double TE104cavity caused an additional deformation of the signal intensity for a 30-mm sample which was moving in the first cavity. The primary effect was that the plateau was replaced by a region in which the intensity increased linearly with sample position,r= 0.99. Each of the above phenomena may be a source of significant errors in quantitative EPR spectroscopy. Cylindrical samples to be compared should be of identical length and internal diameter. Accurate and precise positioning of each sample in the microwave cavity is essential.

On the reaction of ferric heme proteins with nitrite and sulfite.

Optical and EPR spectroscopy of ferric heme proteins of the porphyrin, oxyporphyrin, and isobacteriochlorin classes has indicated that nitrite reacts with these proteins at the heme iron. Sulfite has been conclusively proven to react only with proteins containing the isobacteriochlorin macrocycle. Quantitative EPR spectroscopy of these nitrite and sulfite adducts showed that most contained a substantial quantity of undetectable heme. It is suggested that protein-induced autoreduction of nitrite (but not sulfite) and a strained and/or uniaxial g-tensor are the principal ways by which the silent state is produced.

High and low reduction potential 4Fe-4S clusters in Azotobacter vinelandii (4Fe-4S) 2ferredoxin I. Influence of the polypeptide on the reduction potentials.

Azotobacter vinelandii (4Fe-4S)2 ferredoxin I (Fd I) is an electron transfer protein with Mr equals 14,500 and Eo equals -420 mv. It exhibits and EPR signal of g equals 2.01 in its isolated form. This resonance is almost identical with the signal that originates from a "super-oxidized" state of the 4Fe-4S cluster of potassium ferricyanide-treated Clostridium ferredoxin. A cluster that exhibits this EPR signal at g equals 2.01 is in the same formal oxidation state as the cluster in oxidized Chromatium High-Potential-Iron-Protein (HiPIP). On photoreduction of Fd I with spinach chloroplast fragments, the resonance at g equals 2.01 vanishes and no EPR signal is observed. This EPR behavior is analogous to that of reduced HiPIP, which also fails to exhibit an EPR spectrum. These characteristics suggest that a cluster in A. vinelandii Fd I functions between the same pair of states on reduction as does the cluster in HiPIP, but with a midpoint reduction potential of -420 mv in contrast to the value of +350 mv characteristic of HiPIP. Quantitative EPR and stoichoimetry studies showed that only one 4Fe-4S cluster in this (4Fe-4S)2 ferredoxin is reduced. Oxidation of Fd I with potassium ferricyanide results in the uptake of 1 electron/mol as determined by quantitative EPR spectroscopy. This indicates that a cluster in Fd I shows no electron paramagnetic resonance in the isolated form of the protein accepts an electron on oxidation, as indicated by the EPR spectrum, and becomes paramagnetic. The EPR behavior of this oxidizable cluster indicates that it also functions between the same pair of oxidation states as does the Fe-S cluster in HiPIP. The midpoint reduction potential of this cluster is approximately +340 mv. A. vinelandii Fd I is the first example of an iron-sulfur protein which contains both a high potential cluster (approximately +340 mv) and a low potential cluster (-420 mv). Both Fe-S clusters appear to function between the same pair of oxidation states as the single Fe-S cluster in Chromatium HiPIP, although the midpoint reduction potentials of the two clusters are approximately 760 mv different.

Anaerobic reductive titrations with solid diluted sodium dithionite in an apparatus suitable for EPR spectroscopy

A procedure using sodium dithionite diluted with KCl and small amounts of base has been developed for anaerobic reductive titrations of proteins in conjunction with EPR and light spectroscopy. The reductant is kept dry in a glass capsule until deoxygenation of the protein solution is completed. When 1:100 and 1:300 dilutions of dithionite were tested with solutions of FMN and cytochrome c, the data indicated titers of 88.6 ± 1.7 (S.D.) and 23.7 ± 1.2 (S.D.) nanoequivalents of electrons per mg, respectively. These figures represent recoveries of 92 and 81% of values calculated from the titer of the undiluted dithionite. The random errors associated with such titrations appear to be commensurate with those encountered with quantitative EPR spectroscopy. This procedure has been applied to small (<1 ml) aliquots of concentrated solutions of oxidative enzymes.