Biochimica et Biophysica Acta.

1075(1991) 109-117

109

1991 Elsevier Science Publishers B.V. 0304-4165/91/$03.50

ADONIS 0304416591002316

BBAGEN 23552

A method for preparing analytically pure sodium dithionite. Dithionite quality and observed nitrogenase-specific activities
Charles E. McKenna, William G. Gutheil * and Wet Song
r)epartment of Chemistt3". Unit'ersity of Souther,z Cali]~rnia. Los Angeles, CA (U.S.A.)

**

(Received 1 April Iqgl)

Key words: Sodium dithionite: Recrystallization:Nitrogenase:Redox enzyme:(A. t'ineh~ndii) Sodium dithionite (NazSzO 4) is widely used as a reduetant in biochemical studies, but has not been available in its pure form. A convenient, detailed procedure is given for the recrystallization of commercial dithionite from 0.1 M NaOH-methanol under anaerobic conditions. Twice-recrystallized dithionite had a purity of 99 _+ 1% by I N spectroscopy (A3I s) and elemental analysis. The influence of dithionite quality on the apparent reduction activities of the nitrogenase components (Avl and Av2) from Ay.otobacter vinelandii was investigated.

Introduction
In studies of biochemical systems involving redox-actire species, sodium dithionite (sodium hydrosulfite, Na2S20 4) is ubiquitously employed as a convenient and potent in vitro reducing agent. Dithionite is routinely used in such studies to generate or maintain reduced states of enzymes, electron transfer proteins and coenzymes and to remove traces of dissolved oxygen from solutions required to be anaerobic [1-6]. Dithionite dissociates slightly in aqueous solution at 25C (K~q approx. 1 10 -~ M [2,6-8], kdls approx. 2 s - i [2,9], or higher [7]) forming two equivalents of sulfoxyl radical anion (SO,~). Kinetic evidence has implicated SO~ as the effective electron donor in dithionite reductions of many biologically significant electron acceptors [1-6], including nttrogenase [lO,1l]. Several examples of reductions in which undissociated dithionite ( S , O ~ - ) may also act directly as a reductant have been reported [2,5,6]. A third species, hydrogen sulfoxylate anion (HSO~), has been proposed as the hydride donor in the dithionite reduction of N A D + analogues [12]. The normal oxidation product of dithionite in

* 1986 Moulton Fellow. Current address: P.O. Box 5109. Atomic Energy Institute, Beijing, People's Republic of China. Abbreviations: PC. phosphocreatine;PCK. phosphocreatine kinase. Correspondence: C.E. McKenna. Department of Chemistry. University of Southern California. Los Angeles, CA 90089-0744, U.S.A.

these reactions is sulfite; the dithionite/sulfite couple has been used to poise the reduction potential of aqueous solutions containing redox active species such as flavodoxin and the nitrogenase Fe protein [13,14]. In addition to its biochemical importance, dithionite has long been used as a bleaching agent and continues to find applications in organic [15], inorganic [16], organometallic [17] and radiopharmaceutieal chemistry [tS]. Our present interest in sodium dithionite stems from our ongoing investigations of the nitrogenase enzyme. Sodium dithionite has been the standard electron donor in nitrogenase activity assays, in biochemical and biophysical studies of the component nitrogenase proteins (FeMo and Fe proteins) and in investigations of the FeMo-cofactor [19-25]. D u r i n g purification of nitrogenase, dithionite is normally added to deaerated buffers to help prevent inactivation of the enzyme components by oxygen [26]. In our laboratory a novel property of nitrogenase Fe protein solutions containing dithionite (referred to as "self-oxidation') was previously documented, consisting of the accelerated disappearance of dithionite from such solutions under anaerobic conditions, with attendant oxidation of the protein Fe-S cluster [27-20]. As a prerequisite tt, further investigation of this phenomenon [30,31], we required pure sodium dithionite. In this paper we present analyses of sodium dithionite from four commercial (U.S.A.) suppliers; none of the samples was better than 84% pure. Aqueous solutions of these dithionite samples, even if prepared anaerobically, give acidic solutions. We describe here a prepara-

110 tive recrystallization procedure that affords analytically pure sodium dithionite from commercial material. The effect of dithionite purity on apparent substrate reduction activities in nitrogenase assays is also briefly examined. Methods
Materials Sodium dithionite was obtained from the following suppliers: J.T. Baker Chemical Co., Phillipsburg, N J; Fisher Scientific, Santa Clara, CA; Alfa Products, Danvers, MA; and Sigma Chemical Co., St. Louis, MO. The first two samples were purchased prior to 1984; the latter two were purchased in 1985 and 1986. The Baker sample had been aerobically stored in the laboratory over a lengthy period before testing. Other samples were stored anaerobically and tested soon after purchase. Deionized H 2 0 for 0.1 M N a O H solutions was de-gassed (boiled) before use. Methanol was reagent grade. Cylinder N2, 'prepurified grade', and argon gases were freed of contaminating 0 2 by sparging through a solution of 0.5 M Cr2(SO4) 2 in 0.5 M H2SO 4 over Zn or, more conveniently, by passage through a bed of activated Ridox (Fisher). Solutions referred to as 'deoxygenated' were placed in a vessel which was thrice evacuated and refilled with N 2 or argon using a manifold. General UV spectral measurements were made on a Cary 17 double-beam spectrophotometer (Varian Instruments). Inert atmosphere operations used an N2-filled DriTrain glove box equipped with an O 2 monitor (Vacuum Atmospheres). 0 2 levels in the glove box were maintained at < 1 ppm. Elemental analyses were performed by Galbraith Laboratories, Knoxville, TN. UV spectroscopic analysis o f dithionite solutions Analyses were performed in triplicate or quadruplicate. For each analysis, 110+ 10 mg of sample was weighed into a 50 ml volumetric flask (previously flame-dried under vacuum) and the weight was recorded to the nearest 0.1 mg. The volumetric flask was stoppered with a septum, pumped and refilled with N z three times, pierced by a vent needle and transferred into the inert atmosphere box together with: (1) 20 mg of sodium dithionite in a small vial (required for the preparation of the stock dithionite solution described below); (2) a 100 ml volumetric flask for each analytical sample; (3) a 1.00 ml volumetric pipet; and (4) a cylindrical Suprasil quartz cuvet (1.000 cm pathlength) having a female ground glass neck sealable with a new, tight-fitting rubber scptum stopper. Two solutions were next prepared in the glove box: (1) Stock dithionite, prepared from 20 mg of sodium dithionite

dissolved in 2 ml of stock Tris (deoxygenated 50 m M Tris, pH 9.0); and (2) stock sample buffer (175 ml per sample): for each 1 ml required, 1 tzl stock dithionite was added to 1 ml stock Tris. The analyses were carried out as follows. The cuvet was filled with stock sample buffer, fitted with a septum stopper, removed from the glove box, and the absorbance was measured at 315 nm (A3~ 5 value). If this value did not fall between 0.2 and 0.6 A, the concentration of dithionite in the stock sample buffer was adjusted by adding more stock dithionite or diluting with stock Tris as required. The cuvet was emptied, rinsed with H z O and MeOH, allowed to dry, and returned to the glove box. The sample to be analyzed was meanwhile dissolved to exactly 50.0 ml in stock sample buffer, creating the stock sample solution, and a 100 ml volumetric flask was partially filled with the same reagent. The 1.00 ml volumetric pipet was flushed with stock sample buffer, first from the main reservoir, then from the 100 ml volumetric flask. The curet was filled with stock sample buffer from the 100 ml volumetric flask, the blank A3z5 value was measured and the cuvet was cleaned and returned to the glove box. A 1.00 ml aliquot of the stock sample solution was pipetted into the 100 ml volumetric flask, diluted to the mark with stock sample buffer and the A3t 5 value of the resulting analytical sample solution was measured. This procedure was repeated for each of the remaining samples, and sample purity was then calculated from the Beer-Lambert law using an assumed Emax of 8000 for dithionite [1]: % purity = A3is(sample-blank) x 10 881 mg sample

Sodium dithionite recrystallization The recrystallization appa, atus is shown in Fig. la (schematic) and lb (photograph). All ground glass joints are 24/40 standard taper. The crystallization vessel A * is a flat-bottomed 1000 ml spherical flask modified with a stopcock (A-l), joined via connector B to filtration vessel C. B is made from two male ground glass joints and has an internal lip (lower end as shown in Figs. 1 and 2) to cause liquid introduced through stopcock C-1 to drop directly into A, rather than flow down its inner surface. C is constructed from a fritted glass filter funnel (6 cm diameter, F porosity) modified

* Important safety note: unless this vessel is specially constructed to withstand complete evacuation, a round-bottomed flask (or modified Erlenmeyer-typefilter flask) rated to - 100 kPa must be used instead for safety reasons (due to the hazard of implosion). The same warning applies to vessels F and (3. Standard precautions (taping of vessel bulbs, safety shield) should be taken during evacuation and other procedures involvingthe apparatus.

111 o
Vacuum

\\

~S
Fig. 1. (a) Schematicrepresentation of apparatus for sodium dithionitc recrystallization.See Methodsfor specificationsof individualcomponents. (b) Photograph of assembledapparatus for sodium dithionite racrystallization(cf. a).

by extension to a length of 18 cm (from frit C-2 to the lower ground glass joint), and by addition of stopcocks (C-1, C-3) and ground glass joints as shown. Rubber septum stoppers of appropriate size are secured by wire to the end of C-1 and similarly to the mouths of reservoir flasks F and G when filled (see below). Connection piece D (with vacuum take-off D-l) together with the 2000 ml round-bottomed flask E form the filter flask unit at the top of the apparatus. Evacuation and refilling of apparatus components is accomplished with the v a c u u m / N 2 manifold H via stopcocks H-l, H-2, H-3 and H-4, through 1 / 4 inch x 1 / 2 inch Tygon tubing. Stopcocks A-l, C-l, F-l and G-I are 4 mm Kontes High Vacuum Valves (I(,-826600) with Teflon plunger and sealing surfaces, refitted with Kontes ethylene-propylene O-rings (K-758292-0010). Stopcock C-3 is the 8 m m version of the same Kontes high vacuum valve. Stopcocks H - 1 / H - 4 are standard glass three-way, high vacuum stopcocks. The cannula shown connecting C to F is a 61 am, 18 gauge, double-tipped stainless-steel needle (Aldrich Chemical Co.). The recrystallization procedure is as follows. Flask A is charged with 60 g of sodium dithionite, a magnetic stir bar is added, and assembly with the other components of the apparatus is carried out as depicted in Figs. I and 2 (cannula not inserted), using Dow Corning (Midland, MI) high vacuum grease on ground glass fittings and strong rubber bands to secure components under positive pressure. The apparatus is alternately evacuated and refilled four times with N 2 using H-l and H-2. The upper and lower portions of the appara-

tus are isolated by closing C-3. Reservoirs F and G are then filled with 200 ml of 0.1 M N a O H and 440 ml of M e O H / 0 . 1 M N a O H (80: 20, v/v), respectively, and securely closed with septum stoppers (wired). The solutions are deoxygenated by four evacuation/N2 filling cycles (see safety footnote), accompanied by vigorous agitation, then preheated to 68-70C (water bath). While F is kept under positive N 2 pressure, the cannula is inserted, allowed to flush (N_,) for a few seconds, then connected to C via the septum stopper at C- 1. With C- l closed, a slight vacuum is applied through A-1. Flask A is immersed in a water bath at 6 8 - 7 0 C and C-l is opened, transferring the aqueous NaOH solution from F to A (magnetic stirring). When the transfer is completed, positive pressure is applied to the apparatus, C-1 is closed and the cannula is removed from C and F. Once a homogeneous solution is attained, the cannula is inserted at G-l, allowed to flush, reinserted at C-I and used to transfer the MeOH solution in G into A over a period of 5 min (continued stirring). A white precipitate begins to form once approx. two-thirds of the MeOH solution has been added. At the end of this second addition, stopcock A-1 is closed, flask E is pumped out through H-l, and the entire apparatus is inverted. Positive pressure is applied via A-l, H-1 is closed and C-3 is opened, resulting in filtration of the recrystallized solid. H- 1 is opened to vacuum as required to maintain the flow of filtrate into E. Once filtration is complete (care itaviiig been taken to remove as much residual mother liquor as possible), C-3 is closed. Anhydrous MeOH (300 ml) is

112 then deoxygenated in reservoir G as described above. A is reconnected to G-I with the cannula and approx. 100 ml of MeOH is passed in from G. The precipitate is slurried and the MeOH is removed by filtration via C-3. After repeating this MeOH wash twice, C-3 and C-1 are closed, G is disconnected, E is emptied and vacuum is applied via A-1 until the solid is completely dry (approx. 1 h), with occasional shaking to speed the process. To repeat the recrystallization procedure, the apparatus is carefully returned to an upright position, causing the dithionite powder and stir bar to fall back into A. E is reattached and the procedure is repeated using solvent volumes scaled by a factor of 50-60%. Once the final product is dry, It is transferred into the glove box and weighed. The product should be stored anaerobically in a gas-tight container; normally the sealed storage bottles were kept within sealed larger bottles in a glove-box under N 2. unless this parameter was being examined as a source of error, when no correction was made (see below). Assay mixtures were prepared by aerobically dispensing into each assay bottle 0.4 ml of the HepesATP-PC-PCK reagent and sufficient 25 mM Hepes buffer to give a final volume of 1.00 ml (including dithionite and protein). The bottles were sealed with rubber septum stoppers, then evacuated and filled with argon (four times). To each was then added 0.25 ml of the sodium dithionite assay reagent. After venting, 1.0 ml of C2H2 was injected with a gas-tight syringe and the bottles then revented to the atmosphere. Avl solution was added (amounts given in the figures) to each bottle and after preincubation at 30C for 5 min (shaker bath), assays were initiated by injection of Av2 solution (amounts given in the figures). Assays were terminated after 10 min at 30C by addition of 0.25 ml 25% trichloroacetic acid. Reduction product C2H4 and substrate C2H2 were quantitated on a 100 x 2.5 mm column of Porapak N using a Varian 1440 gas chromatograph with flame ionization detection. The chromatograms were recorded and integrated using a HewlettPackard 3390A Integrator. C2H 2 was used as an internal standard for CzH 4 formation [35]. The effect of using dithionite of varying purity in nitrogenase activity assays was determined by titrating each protein with its complement to define both the maximal activity of the titrand component and the form of the activity titration curve. For unpurified dithionite samples, the concentration of stock solutions was corrected to reflect the actual dithionite content. The results were then compared to activities obtained using the nominal dithionite concentration.

Nitrogenase assays
The nitrogenase components from A. ~'inelandii (Avl and Av2) were prepared as described elsewhere [32]. Avl and Av2 solutions (in 25 mM Hepes, pH 7.4) were stored as frozen pellets in LN2 and thawed anaerobically when needed, with addition of 2%, v/v, of 100 mM dithionite/25 mM Hepes. The Avl and Av2 specific activities (SA values, nm C2H2 reduced to C2H4/mg limiting protein per min) of components used in particular experiments are specified in the Results or in the corresponding figures. All assays were performed under argon in 21.5 ml rubber-stoppered vaccine bottles containing 1 ml of assay reaction mixture and 1 ml of C2H 2. C2H 2 (high purity grade, MG scientific gases) was passed through a dry iee/isopropanol trap to remove acetone before use. The assay mixture contained dithionite (vat.), 5 /.tmol ATP (Sigma), 25 ttmol PC (Sigma), 7.8 units PCK (Sigma), 5 /~mol MgCI2.6H20 (Baker), and 25 /xmol Hepes buffer (Sigma) at pH 7.4. The concentrations of Avl, Av2 and dithionite for specific experiments are indicated in the Results or in the appropriate figures. Avl and Av2 molar concentrations were calculated using molecular weights of 220000 and 63000, respectively [33], from uncorrected protein concentrations measured using the Biuret method [34] with bovine serum albumin as standard. No attempt was made to normalize molar concentrations on specific activity, hence A v l / A v 2 ratios corresponding to maximal activities in different experiments are empirical. Sodium dithionite reagent solutions for assays (0.1 M) were prepared (glove box) in 25 mM deoxygenated l-lepes (pH 7.4; the pH was readjusted to 7.4 if necessary). In certain experiments, the dithionite salt was dissolved in unbuffered H 2 0 containing 0.02 M NaOH (Results). The dithionite concentration in nitrogenase assays was standardized at 25 mM and adjusted for reagent purity,

Results

Dithionite analysis by UV absorbance

To measure the purity of commercial sodium dithionite and. initially, of our reerystallized material, we relied upon the emax value established by Dixon using spectrophotometric titration against ferricyanide [1] (see Discussion). However, in preference to the bench-top anaerobic techniques for sample preparation detailed by this author, we found the glove-box procedures described in Methods to be simpler and more convenient. Reproducibility data for four analyses on the same Na2S204 sample are given in Table 1. The normalized standard error of the mean (0.3%) is comparable to the expected error of the UV absorbance measurement.

Purity of commercial sodium dithionite

The Fisher, Sigma and Alfa samples were stored under an inert gas immediately after unsealing the original containers. All three samples were free-flowing, clump-free white powders having little or no odor,

113 TABLE I
Single-sample UP" absorbance analytical data for twiee-recrystallized sodium dithionite a Dithionite purification U V analysis d a t a for r e c w s t a l l i z e d s o d i u m d i t h i o n i t e s a m p l e s a r e p r e s e n t e d in T a b l e II. R e g a r d l e s s of the c o m m e r c i a l s o u r c e o f t h e sample, 9 3 - 9 6 % p u r e m a t e rial w a s o b t a i n e d in o n e pass. T h e p u r i t y level a c h i e v e d did n o t a p p e a r to d e p e n d g r e a t l y o n the initial quality of the s a m p l e ( c o m p a r e Expt. 2 with Expts. 1 a n d 3 - 7 ) . A s e c o n d recrystallization usually b r o u g h t the p u r i t y to 9 9 - 1 0 0 % , at w h i c h level f u r t h e r i m p r o v e m e n t f r o m a t h i r d recrystallization b e c a m e difficult to d e t e c t by U V absorbance. T a b l e III s h o w s e l e m e n t a l analysis d a t a (Na, S, C a n d H to d e t e c t c a r b o n a t e a n d r e s i d u a l solvent) for several o f the recrystallized samples. U s i n g a n e a r l i e r version o f o u r s t a n d a r d p r o c e d u r e ( n o t e d, T a b l e II), a n d s t a r t i n g f r o m t h e d e t e r i o r a t e d B a k e r s a m p l e , low N a a n d S p e r c e n t a g e s ( N a , - 1.28%; S, - 1.59%) w e r e d e t e r m i n e d for the 2 recrystaUized m a t e r i a l a n d a t h i r d recrystallization w a s n e e d e d to achieve analytically p u r e m a t e r i a l (Na, - 0 . 0 9 % ; S, + 0 . 0 4 % ) . A s n o t e d above, t h e s e 2 a n d 3 recrystallized s a m p l e s w e r e n o t readily d i s t i n g u i s h e d In p u r i t y by U V abs o r b a n c e analysis ( T a b l e 11, Expt. 2). E l e m e n t a l analysis f o r N a a n d S t h u s a p p e a r e d to b e m o r e sensitive to r e m a i n i n g i m p u r i t i e s in t h e s e reerystallized d i t h i o n i t e samples. Starting from a 'good' commercial sample, analytically p u r e m a t e r i a l w a s o b t a i n e d u s i n g t h e s t a n d a r d p r o c e d u r e ( M e t h o d s ) a f t e r o n l y t w o recrystallizations (Na, - 0 . 1 1 % ; S, ~-0.02%) (Expt. 6). In b o t h cases, the s t a n d a r d e r r o r f o r d u p l i c a t e a n a l y s e s w a s e q u a l to o r g r e a t e r t h a n t h e d i f f e r e n c e s b e t w e e n t h e e x p e r i m e n t a l a n d t h e o r e t i c a l values.

Sample wt. (mg) 107.6 101.3 110.7 100.4

Blank UV (,431s) 0.205 0.250 0.232 0.260

Sample UV (A :,is) 1.202 1.190 1.255 1.181

AUV Sample wt./ ( A .~t~) AUV t, 0.997 107.9 0.940 107.8 1.023 108.2 0.921 109.0

Samples diluted as described in Methods prior to UV measurements. " Mean 108.25:0.3 S.E.

c h a r a c t e r i s t i c s w h i c h a r e usually a s s o c i a t e d with minimal decomposition. These samples thus represent ' g o o d ' c o m m e r c i a l s o d i u m d i t h i o n i t e , s t o r e d to minimize d e c o m p o s i t i o n f r o m o x y g e n e x p o s u r e . T h e B a k e r sample, which was lumpy, non-free-flowing and malodorous, represents commercial sodium dithionite m a d e ' b a d ' by n o n o p t i m a l s t o r a g e over a p r o l o n g e d period. UV analytical data for the commercially obtained s a m p l e s a r e given in the ' 0 ( z e r o times) recrystallized' c o l u m n o f T a b l e II. D i t h i o n i t e c o n t e n t in the ' g o o d ' s a m p l e s (Expts. 1 a n d 3 - 7 ) r a n g e d f r o m 78 to 8 4 % , r e p r e s e n t i n g the b e s t p u r i t y available w h e n suita b l e p r e c a u t i o n s a r e t a k e n to avoid d e c o m p o s i t i o n d u e to oxygen exposure. The 'bad' air-stored sample analyzed at only 5 2 % d i t h i o n i t e (Expt. 2), c o n s i s t e n t with t h e visual a n d o l f a c t o r y e v i d e n c e o f its d e t e r i o r a t i o n .

TABLE II
I.]l/ analytical data Jor commercial and recrystalhzed sodium dithionite

The purity of the samples were as follows: Expts. !-4, duplicate values, 5:0.5% S.E.: Expl. 5, data from Table I; Expts. 6, 7, 0.2%, 0.6% S+E, n = 4. For details of sample origin see Methods, The 'Sld.' analytical method was that described in Methods. AI was a less rigorous procedure, in which a sodium dithionite sample (50 rag) was weighed into a 50 ml volumetric flask, covered with perforated parafilm, transferred into the glove box (02 < 1 ppm) and dissolved in 25 mM deoxygenated Hepes buffer (pH adjusted to 9.0-9.2 with NaOH). An aliquot of this solution was placed in a gas-tight Suprasil quartz cell (10 mm path length) which was closed with a new rubber septum stopper and removed from the glove box for spectroscopic measurements. This method generally gave the same results as the standard method ::1:0.5%(cf. Expts. 3, 5-7 (0)) but was not as reliable. Under Rxtal method, the "Std." method is that described in Methods. except that in Expt, 7 a third in sitn recrystallization step was added. RI was an earlier version of the standard procedure, in which sodium dithionite (30 g) in 138 ml of O2-free 0.1 M NaOH (66-68C) was treated with 162 ml of 0.1 M NaOH in 90% aq. MeOH (66-68C. 5-10 min); after filtration, the product was washed (3 ) with 90% McOH and dried ( < I ram) over KOH for 8-12 h. Additional recrystallizations were done in the same way, with solvent volumes scaled to product yields. Expt. No. I 2
3 4

Sample origin Fisher Baker

Sigma

Analytical method AI AI
Al

Rxtal. method RI R1
R!

Purity (%) recrystallized:

OX IX 2x 3X

78 52
81

93 95.5
94

99 99
97

99
-

5 6 7

Alfa Sigma Sigma Sigma

AI Std. Std. Std.

RI Std. Std. Std.

84 81 81 81

92.5

98 100.5 99 -

99

114 TABLE 111

Elemental analytical data for recrystallized sodium dithionite A v l C e l l 2 reduction activities

Sample origin Baker +' 1 Rxt. 2X Rxt. 3x Rxt. Sigma~ 2 Rxt. Theory

Rxtal. method

Elemental analysis (%)

RI Rl RI Std.

0.l 0.1 <0.1 < 0.1 0.0

0.2 <0.1 <0.1 < 0.1 0.0

24.43 25.13 26.32t' 26.30e 26.41

33.69 35.24 36.87c 36.85f 36.83

~' Correspondsto Expt. 1 from Table II. h +0.11% S.E., n=2. +0.17% S.E., n=2. a Correspondsto Expt. 6 from Table 11. +0.10% S.E., n = 2. t :t:0.19% S.E., n = 2.

Titration of limiting Avl (0.23 tiM) with Av2 (spec. act. 1750, 3-12 /*M) generated the expected Michaelis-Menten-like saturation curve, defining a Vm,x (spec. act. 1900) for Avl when [Av2] >> [Avl] (data not shown). When double-reciprocal specific activity replots were constructed for titrations of Avl by Av2 using 98% and fresh commercial (Alfa, 84%) dithionite, essentially the same ( + 1%) Vma x value (spec. act. 1900) was obtained when a concentration correction was made for the reagent purity (data not shown). Triplicate determinations at two separated A v 2 / A v l ratios were used following the recommendation of Endreny [36]. Similar results were obtained when single activity points at 5-6 different A v 2 / A v l ratios were determined. When the experiments were repeated using nominal (unadjusted) initial concentrations of 25 mM dithionite, again no significant difference in Vm,x was observed (data not shown).
A v 2 C e l l 2 reduction activities

On one recrystallization, under the standard conditions, 60 g of commercial (81%) material gave 30.9 g (63%) of 93-94% pure product (one run gave 31.9 g (66%)), which after the second recrystallization gave 15.9 g (33%) of analytically pure sodium dithionite. An additional (third) recrystallization gave an overall yield of 9.7 g (20%), but no change in purity could be detected within the error of the UV analysis. Commercial sodium dithionite is inexpensive, therefore conditions were adjusted to optimize purity rather than yield. The products from Expts. 5-7 in Table 11 (referred to as '99% dithionite' in the enzyme activity studies presented below; stored samples assaying at 98% when used are so identified; see Discussion for further comments on purity) were free-flowing, white microcrystalline solids having negligible odor. When dissolved in deoxygenated H 2 0 anaerobically, they produced neutral solutions. Purified dithionite stored in a sealed glass bottle within the glove box at room temperature decomposed by 1% or less over 2 months. Our standard recrystallization procedure has several advantageous features. Two (or more) sequential recrystallizations are 'telescoped' without isolating the intermediate (1 or 2 x recrystallized) product, and the anhydrous MeOH wash facilitates drying: analytically pure material is afforded in adequate yield in a single day's operation. If the product is isolated, dried, and analyzed after each recrystallization, 4 days of operations are required for three recrystallizations. It should be noted that the teflon stopcocks of the apparatus shown in Figs. 1 and 2 are considerably more convenient for use with alkaline aqueous MeOH solutions than all-glass stopcocks, provided that the recommended retrofit with the more solvent-resistant ethylene-propylene O-rings is done.

Titration of limiting Av2 with Avl normally produces a plot showing a maximum at an A v l / A v 2 ratio of 1-2; at higher ratios, Av2 is increasingly inhibitory. Av2 (spec. act. 1750 or 1650, concn. 1.19/~M) titrated with Avl (spec. act. 1840, variable concentration) using rccrystallized (98%) vs. commercial (Alfa, 84%) dithionite as electron donor at 25 mM had virtually indistinguishable maximal activity A v l / A v 2 ratios (1.5) and activity maxima (difference 2-3%), for either true or uncorrected dithionite concentrations (data not shown). If the dithionite titer was decreased to 50% (uncorrected) by mixing almost completely deteriorated reagent (8%) with fresh commercial (Alfa, 84%) dithionite, while maintaining the reagent pH at 7.4, the maximal activity fell by 20% and occurred at a 10% lower A v l / A v 2 ratio (Fig. 2). In a similar experiment,

0 E '

l.oo
2O 4o 50 6o

Avl (uL)

Fig. 2. Av2 (I.2 v.M) specific activity as a function of added Avl (45.5 p.M, spec. act. 1770). Nominal assaydithionite concentration = 25 raM, uncorrected for dithionite purity; dithionite stock reagent prepared in 25 mM Hepes (pH 7.4). Legend: A, data for 98% dithionite; O, data for 50% dithionite (mixture:see Results).

115
,.4-

i
T~ ca E <

20

4.0

~0

~I0

Avl (uL)

Fig. 3. Av2 (3.0 p.M) specificactivityas a function of added Avl (125 p.M, spec. act. 2220). Nominal assay dithionite concentration = 25 mM, uncorrected; pH adjustment of dithionite stock reagent, prepared in H20, using 1 M NaOH (0.8 ml/40 ml reagent). Legend: 4. 99@/~ dithionite; o, 95% dithionite (1 recrystallized); II, 78% dithionite (Fisher); , , 52% dithionite (Baker, deteriorated).

we compared 99%, 1 x recrystallized (95%), fresh commercial (Fisher, 78%) and deteriorated commercial (Baker, 52%) dithionite reagents, prepared in H z O and 'neutralized' using 1:40 (v/v) of 0.8 M NaOH without verification of pH. Significant variations in both titration parameters were observed among these samples (Fig. 3).

Discussion
Sodium dithionite has not been available from commercial suppliers as an analytical reagent, and until recently was offered by most manufacturers without accompanying purity data. Noting that it was not practicable to make up a solution of known concentration by weighing out the solid, which "usually gives appreciably less than the theoretical concentration", Dixon determined a nominal ~,,~, of 8000 M - t c m -I for sodium dithionite standardized by titration of a chromophoric oxidant [1]. Using this value, we found freshly purchased commercial samples from three vendors to have dithionite contents ranging from 78% to 84%. Commercial material for which no special storage precautions had been taken over a prolonged period ( > 1 year) was only 52% pure. Samples of dithionite analyzing in the 90-96% range have occasionally been referred to in the literature [2,6,13]. In a small number of studies carried out in the 1970s, nearly pure sodium dithionite obtained in limited amount from a particular manufacturer (Associated Chemicals Co., Ltd., U.K.) is mentioned [11,37]. Unfortunately this company, then known to a few cognoscenti, no longer exists (G..I. Leigh, personal communication), and the unavailability of 'analytical grade' dithionite has continued to cited as an obstacle to some uses of the reagent [38].

Sodium sulflte is known to be a major impurity in commercial sodium dithionite. In a study of the effect of redox potential on nitrogenase activity, Na2S204 from Merck was found to contain 20% sulfite [14]. Mayhew [13] has pointed out that the redox potential of dithionite solutions becomes more positive as the SEOj - concentration is increased, if a few percent of contaminating SO~- is present. This may result in inadvertent oxidation of a low-potential electron acceptor, when excess dithionite is added with the intention of ensuring reduction. Sulfite also affects the rate of nitrogenase-catalyzed $ 2 0 ~ - disproportionation, accelerating it at low levels, but retarding it at higher concentrations [30,31]. Efforts to remove sulfite impurities from dithionite reagents by precipitation with Ba 2 have not been fully satisfactory [12]. Additional impurities may include other sulfur compounds and sodium carbonate. We have not found any comprehensive published information on possible trace contaminants, such as transition metal ions, in commercial sodium dithionite. A number of authors have reported lack of success in attempts to purify sodium dithionite by recrystallization to homogeneity, both in the older literature [39] and more recently [40]. A patent method claiming preparation of granular sodium dithionite of 95% purity from aqueous M e O H solutions at pH 11-12 has been available for two decades [41]. In the example given in the patent abstract [41], the starting dithionite appears to have been 80% pure overall, but already 95% pure relative to Na2SO 3 (the remaining impurity cited was Na2CO3). Repeated precipitation of commercial dithionite using a modification of this method was recently reported to give solids containing up to 90% dithionite by U V analyses [42]. To assess the purity of dithionite recrystallized by our methods, we needed an accurate determination of its concentration. Titrative reagents based on UV-visihie spectroscopy previously used to standardize dithionite solutions include ferricyanide ion (Fe(CN) 3-) [1,4], lumiflavin 3-acetate [2,3], methylene blue [40] and FMN [43]. Indirect methods such as measurement of sulfite content [14], if used alone, rely upon the assumption that no other contaminant is present and are therefore inherently less reliable. We initially chose direct UV spectroscopic analysis using the dithionJte max of 8000 M - t c m -~ measured by Dixon at 314 nm (the samples were dissolved in 0.2 M phosphate buffer (pH 7.6)) [1]. This number was obtained by rounding the average of four results from ferricyanide standardizations, which gave a purity of 96% for the sodium dithionite sample used. The actual mean from Dixon's data is 8006 M -I cm - t +58 S.E.; its accuracy depended on the certainty with which the molar absorbance of ferricyanide was known, stated to be within + 1% [1]. Later workers have taken the Am,x of

116 dithionite to be 313 nm [41], 315 nm [13] and 320 n m [6]; in our work a ~rnax of 315 nm was found. Our purest samples were recrystallized to constant molar absorbance and analytical (Na, S) purity. These samples, estimated to contain < 2% Na2SO 3 (if formed by oxidation: isoproportionation of dithionate to (2) sulfite and thiosulfate e.g. would not affect % N a / % S results) and < 0.1% Na2CO 3 by elemental analysis, analyzed at 98-100.5% using the Emax= 8000 M -I e m - ~ value. If 100% purity is assumed, the e3~5 of the twice-recrystallized material from Expt. 6 in Table II is calculated from the data in Table I to be 8043 M cm -~ +21 S.E. Although the dithionite emax value reported in this work might be preferred on the basis that it was determined using a purer sample, there is not a highly significant difference between our value and Dixon's. The availability of highly purified dithionite has been instrumental in the elucidation of Av2 'self-oxidation', as reported elsewhere [30,31]. In this paper we have cursorily examined the effects of dithionite quality on A. vinelandii nitrogenase activity assays. We adopted the standard assay dithionite concentration of 25 m M to facilitate comparisons, although this may not be quite optimal for all conditions (W. Gutheil, unpublished data). Three conclusions can be drawn from the results. Firstly, improvement in Avl activity from using the pure reagent instead of fresh commercial dithionite (84%) is negligible, even if no correction for the actual dithionite content is made, provided that the reagent pH is properly adjusted and the Av2 concentration is saturating. Secondly, although at most a small increase in Av2 specific activity ( < 3%) is obtained using 2 x recrystallized dithionite in place of fresh commercial reagent when the dithionite stock reagent pH is adjusted to 7.4 and buffered, a significant underestimation of Av2 specific activity occurs when deteriorated dithionite is used. These results are reasonable, given the assumption that the impurity in the dithionite samples is (mainly) sulfite, and the fact that the S 2 0 ~ - / H S O ~ ratio sets the redox potential of the nitrogenase assay solutions. Dependence of Azotobacter nitrogenase activity on the solution potential was demonstrated previously [14]. The effect should be more pronounced in Av2-1imited than in Avl-limited assays because in nitrogenase turnover, it is known that electrons flow from dithionite to Av2 and thence to Avl. U n d e r the particular nitrogenase assay conditions employed in this work (A. vinelandii enzyme, assay pH of 7.4, etc.) it would appear that when >_ 84% dithionite is used without a titer correction, the effect on Av2 activity is rather small at the detection level of a standard assay. However, below this threshold, as in commercial samples that have deteriorated during storage, significant underestimation of the Av2 activity is possible. In systems having a smaller separatio n between the enzyme and dithionite reduction potentials, the threshold would be expected to be higher. Thirdly, when the pH of dithionite stock solutions used for Av2 assays is adjusted arbitrarily with a fixed amount of NaOH determined for one particular dithionite sample, samples of differing purities may display altered Av2-Avl titration curves due to a combination of dithionite concentration and assay pH effects, possibly resulting in significant underestimation of Av2 activity. Such errors may also arise if the dithionite sample for which a neutralizing equivalent of N a O H has been determined, subsequently deteriorates. It would appear simplest and most prudent to utilize pure dithionite in studies of nitrogenase components (and redox enzymes generally), thereby eliminating all potential errors due to underestimated concentration, increased solution potential and the effects of acid or other possible contaminants. In conclusion, the results presented here demonstrate a reliable procedure for preparation of analytically pure dithionite. The availability of this material should prove of value in many biochemical and chemical applications of dithionite.

Acknowledgement
We thank Professor Philip J. Stephens for the use of his Cary 17 spectrometer, and Mr. James Merritt for glassblowing work. This research was supported by U S D A ( C R G O (82-CRCR-l-l106)), NIH (GM-31716), and the Herman Frasch Foundation (HF-67).

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