ANALYTICAL

BIOCHEMISTRY

190, 348-353

(1990)

The Small-Scale Production of [U-‘4C]Acetylene from Ba14C0,: Application to Labeling of Ammonia Monooxygenase in Autotrophic Nitrifying Bacteria Michael

R. Hyman

and Daniel J. Arpl

Laboratory for Nitrogen Fixation Research, Department of Botany and Plant 4100 Cordley Hall, Oregon State University, Corvallis, Oregon 97331

Received

April

13, 1990

A small-scale method has been adapted from an established procedure for the generation of [U-14C]acetylene from inexpensive and commonly available precursors. The method involves the fusing of Ba”‘CO, with excess barium metal to produce Ba14Ca. The BaC, is reacted with water to generate acetylene which is then selectively dissolved into dimethyl sulfoxide (DMSO). The results presented demonstrate the effect of Ba:BaCO, ratio on the concentrations of various gases released during the hydrolysis reaction and quantify the selectivity of the DMSO-trapping process for each gas. [U-14C]Acetylene generated by this method has been used to inactivate ammonia monooxygenase in three species of autotrophic nitrifying bacteria: Nitrosomonas europaea, Nitrosococcus oceanus, and Nitrosolobus multiformis. Our results demonstrate that acetylene inactivation of this enzyme in all three species results in the covalent incorporation of radioactive label into a polypeptide of apparent M, of 25,000-27,000, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. o 1990 ACMMIC PW,, I~C.

Acetylene (C!,H,) is known to inhibit several metalloenzymes, including, but not limited to, nitrogenase, nitrate reductase, nitrous oxide reductase, hydrogenase, cytochrome P-450, and ammonia and methane-oxidizing monooxygenases (4). For all the enzymes listed above, acetylene is thought to interact at the metal-contaming active site of the target enzyme, although the mechanisms of inhibition and inactivation vary considerably. Although acetylene is potentially a versatile compound for mechanistic studies and stoichiometry de-

1 To whom 348

Pathology,

correspondence

should

be addressed.

terminations, little use has been made of this inhibitor in biochemical studies of sensitive enzymes. Recently, we have been using the very specific and irreversible inactivation of ammonia monooxygenase by acetylene in studies of ammonia oxidation in autotrophic nitrifying bacteria such as Nitrosomonas europaea. For both ammonia and methane monooxygenase, acetylene acts as a suicide substrate which inactivates the enzyme through covalent attachment to the target enzyme (6,B). This process results in the incorporation of radiolabel from [*4C]acetylene into a membrane-bound polypeptide with an approximate 1M, 28,000 (6). The specificity of this reaction and the use of [‘“C]acetylene provides a powerful tool to aid in the purification of this enzyme. Our study of ammonia monooxygenase and studies of other acetylene-sensitive enzymes would be greatly enhanced if radioactive forms of acetylene of high specific activity were readily available, economically priced, and supplied in a concentrated and easily manipulable form. Although numerous methods exist for the production of acetylene, the simplest and most well-known procedure is the hydrolysis of metal carbides. One form of radioactive acetylene can be generated by hydrolyzing calcium carbide (CaC,) with tritiated water to produce C,T,. However, since acetylene is a relatively acidic gas (pKa = 22 compared to pK, = 44 for C,H,) (4) it can exchange protons reasonably rapidly with protic solvents and accordingly the usefulness of tritiated acetylene is somewhat limited. A more useful form of radiolabeled acetylene is [U-14C]acetylene which can be prepared by the hydrolysis of metal carbides generated from labeled precursors. However, most commercial suppliers do not offer either radiolabeled carbides or [14C]acetylene as “off the shelf’ reagents. When available, commercially produced [14C]acetylene is supplied in a gaseous state which readily polymerizes to form an amorphous black compound known as “cuprene” and 0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

RADIOLABELED

ACETYLENE

therefore has a very limited shelf life. A further complication of an expensive commercial custom synthesis of [‘*C]acetylene is that the gas is usually supplied in vials under negative pressure and is therefore difficult to manipulate. To overcome these problems we have adapted and significantly simplified a previously described method to generate [U-14C]acetylene from Ba14C0,. The process described here utilizes readily available and reasonably priced precursors and allows one to generate [U-14C]acetylene as required. A further development is the use of the high solubility of acetylene in dimethyl sulfoxide (DMS0)2 both as a means of purifying and storing the gas. DMSO also provides a convenient liquid medium in which to dilute or manipulate the gas. When dissolved in DMSO, [U-14C]acetylene is stable and reproducible results in our studies of nitrifying bacteria have been obtained after storage of the DMSO/acetylene mixture for 1 year at -20°C. MATERIALS

AND

METHODS

Muteriuls. Barium metal (99%) and unlabeled BaCO, (99.999%) were obtained from Aldrich Chemical Co. Inc. (Milwaukee, WI). Ba14C0, (specific activity 57.5 mCi/mmol) was obtained from Amersham Corp. (Arlington Heights, IL). Electrophoresis materials were supplied by Bio-Rad (Richmond, CA). Gases for gas chromatography and calibrations were obtained locally. Generation of barium carbide (BaCJ and acetylene. In the synthesis of both unlabeled and labeled barium carbide, thin slices of barium metal were chopped from barium rods using a razor blade and the resulting metal flakes were further cut into small fragments (approx 1 X 1 mm). This process was conducted under hexane to limit oxidation of the metal. The metal flakes were then transferred to a Pyrex test tube (5-ml volume) and the residual hexane was evaporated under a stream of CO,-free argon. Finely powdered BaCO, was then placed on top of the metal flakes and care was taken to evenly distribute the powder over the metal without causing the powder to settle to the bottom of the tube. The tip of the tube was then heated in a bunsen flame until the mixture briefly incandesced and set to a black amorphous mass (less than 10 s). The resulting fused Ba and BaC, mixture was then cooled under a stream of CO,-free argon. The tip of the test-tube was broken off and the fused mixture was transferred to a reaction vessel for hydrolysis. The Ba/BaC, fusion mixtures were hydrolyzed in vials which were stoppered with butyl rubber stoppers and aluminum crimp seals and evacuated using a vacuum manifold. The hydrolysis reactions were initiated by adding water to the vials. In all cases the reaction was

2 Abbreviation

used:

DMSO,

dimethyl

sulfoxide.

FROM

Ba’%O,

349

allowed to continue until all gas evolution had ceased and the Ba/BaC, mixture had completely hydrolyzed. The pressure in the vial was increased to 1 atm by puncturing the stopper briefly with a syringe needle to allow air to enter the vial. Trapping of acetylene with DMSO. To obtain acetylene as a solution in DMSO, Ba/BaC, mixtures (either labeled or unlabeled) were hydrolyzed in a stoppered serum vial (14 ml) which had been modified by cementing an open-topped, l-ml vial to the inside base. The Ba/BaC, mixture was placed in the open inner chamber and the reaction was initiated by the addition of water (200 ~1). After gas evolution had ceased and the hydrolysis reaction had reached completion (typically 2 h), 500 ~1 DMSO was injected into the outer chamber of the reaction vial and the vial was then placed on an orbital shaker (75 rpm) for 15 min. The DMSO was then removed by syringe and was replaced with fresh DMSO and the vial was returned to the shaker. This procedure was repeated four times. The DMSO removed after each cycle was injected into a 4-ml stoppered vial and stored frozen at -20°C.

The generation of U-‘4C-labeled acetylene. The carbide-generating reaction mixture consisted of 1 mCi Ba14C0, (3.46 mg) with a specific activity of 57.5 mCi/ mmol which was fused with 70-80 mg barium metal, as described above. The resulting metal/carbide mixture was hydrolyzed with 200 ~1 water in the inner chamber of a double-chambered, stoppered 14-ml vial. After completion of the DMSO-trapping procedure, 1~1 of the 2 ml of the recovered DMSO/[U-‘*C]acetylene solution contained 6.65 X lo5 dpm, as determined by liquid scintillation counting. Gas chromatography. Hydrogen was quantified using a Shimadzu 8A gas chromatograph (Kyoto, Japan) fitted with a thermal conductivity detector, operated at 100°C with a current of 70 mA. A stainless steel column (16 ft X i in) containing molecular seive 5 A was used and was operated at 35°C. Argon was used as carrier gas at a flow rate of 40 ml min-‘. All other gases were quantified using a dual-column Shimadzu 8A gas chromatograph fitted with a flame ionization detector. Methane and ethane were separated and quantified using a stainless steel column (6 ft X $ in.) packed with Porapak Q (So-100 mesh) (Waters & Associates Inc., Milford, MA). Acetylene and ethylene were separated using a stainless steel column (6 ft X $ in.) packed with Porapak N (80-100 mesh). Both columns were operated at 70°C with a detector temperature of 100°C. Nitrogen was used as carrier gas for both columns at a flow rate of 30 ml min-‘. All chromatograms were recorded and analyzed using a Shimadzu C-R3A integrator. Growth of nitrifying bacteria. Cells of N. europaea were grown in l-liter batch cultures as described

350

HYMAN

previously (5). Nitrosococcus oceanus (ATCC 19707) was grown as l-liter batch cultures in 2-liter flasks using a medium described by Watson (9). All medium components for l-liter were added to 900 ml of filtered seawater and this was autoclaved for 20 min. One hundred milliliters of sterile 0.25 M Hepes/NaOH (pH 8.0) buffer in distilled water containing 0.005 g/liter phenol red was also added. Nitrosolobus multiformis (ATCC 25196) was grown as l-liter batch cultures in 2-liter flasks using the medium described by Watson et al. (10). A buffering solution (0.25 M HepeslNaOH (pH 8.0) containing 0.005 g/liter phenol red was also used as described above for N. oceanus. For both N. oceanus and N. multiformis, fresh medium was inoculated with 10 ml from a previous mature culture and new cultures were harvested after full depletion of ammonium, which was typically 5-6 days after inoculation. Harvesting of cells and exposure to labeled acetylene. Mature cultures of all species were harvested by centrifugation at 25,000g for 20 min. The resulting sedimented cells were then resuspended in 50 mM sodium phosphate buffer (pH 7.8) containing 2 mM Mg2+ for N. europaea and N. multiformis or a 9:l mixture of filtered seawater and 0.25 M Hepes buffer in distilled water (pH 8.0) for N. oceanus. The cells were then sedimented again by centrifugation at 25,000g for 20 min and finally resuspended at approximately 0.1 g wet weight/ml of appropriate buffer, as above. Harvested cells were exposed to [U-14C]acetylene by incubating the cells in 30ml stoppered serum vials which contained up to 3 ml cell suspension, and ammonium ions were added to a 2 InM final concentration. An aliquot of the [U-14C]acetylene/ DMSO solution (50 ~1) was also added to each serum vial and the vials were placed on an orbital shaker operated at 120 rpm. After 1 h, the cells were removed from the incubation mixture by centrifugation (25,OOOgfor 20 min) and all species were resuspended at approximately 0.1 g wet weight/ml in 50 InM sodium phosphate buffer, pH 7.8, containing 2 mM Mg2’. The cells were then frozen and thawed three times and centrifuged (25,OOOgfor 20 min), as described previously for N. europaea (6). A sample of the sedimented material (100 pg protein) was solubilized in microfuge tubes in sample buffer containing 1% (w/v) sodium dodecyl sulfate, 10% (w/v) glycerol, 10% (v/v) &mercaptoethanol, and 62.5 mM Tris/ HCI, pH 6.8. The samples were solubilized at 60°C for 3 min and nonsolubilized material was sedimented by centrifugation. Electrophoresis was conducted at room temperature in 13.5% polyacrylamide slab gels. The gels were stained and prepared for fluorography as described previously (6). Fluorographs were produced using Kodak XAR 5 X-ray film and a 5-day exposure time. RESULTS

AND

DISCUSSION

The description of the generation of U-14C-labeled acetylene from a barium metal and carbide mixture on

:--AND

.-AKI’

which our present method is based was provided by Cox and Warne (1). Their method represents the first step in a complex procedure for the generation of labeled ethylene oxide and ethanol (1). The procedure describes the heat-catalyzed fusion of 2 g finely shredded barium metal with 14C-labeled barium carbonate diluted with unlabeled carbonate (total, 1.515 mmol BaCO, containing 1.74 PCi “C). The resulting fusion mixture containing BaC, was then hydrolyzed with water to release the acetylene which was subsequently trapped on charcoal and recovered by vacuum distillation. The procedure resulted in a 137% yield (based on pressure determinations) and a 97% recovery of 14Clabel. For our purposes, the principal drawbacks of this large-scale approach are twofold. First, depending on the ratio of dilution of Ba14C0, with unlabeled BaCO,, there is a concurrent decrease in the specific activity of the acetylene generated by hydrolysis reaction due to the formation of single-labeled and unlabeled barium carbide. Second, the high level of efficiency of carbide formation and subsequent acetylene generation described by Cox and Warne (1) was achieved by using large amounts of barium metal. This efficiency was offset by the large volumes of H, generated (2 g barium will release in excess of 300 ml H,) and an estimated molar ratio of H, generated to 14Cof approximately 4.7 X 106. Such large gas volumes containing low concentrations of low specific activity acetylene are inconvenient to manipulate and store. To remove the contaminating gases present (principally H,), further purification steps are required which are often beyond the capabilities of nonspecialized laboratories. To overcome these problems we have significantly scaled down the method of Cox and Warne (1) to produce small volumes of acetylene with high specific activities and significantly lower accompanying concentrations of H,. The experiment described in Table 1 investigated the effects of our modifications on the efficiency of acetylene production and made use of unlabeled BaCO, as a starting material. Our results (Table 1) demonstrate that efficient generation of unlabeled acetylene from BaC, involves establishing a suitable ratio of barium metal and BaCO, used in the initial carbide-forming reaction. When measured in terms of the amount of acetylene gas generated from a constant amount of BaCO,, there was a progressive increase in the efficiency as the ratio of metal to carbonate was increased. This reflects increases in the efficiency of the fusion process used to generate the BaC, rather than changes in the subsequent hydrolysis reaction which in all cases was observed to reach completion. However, concomitant with the increase in efficiency of acetylene generation, there was also a concurrent increase in the amount of H, generated. Hydrogen generation was proportional to the amount of barium metal added in the fusion reaction and this major gaseous product repre-

RADIOLABELED

ACETYLENE TABLE

FROM

351

Ba14C0,

1

Effect of Barium Metal to Barium Carbonate Ratio on the Generation of Acetylene from Barium/Barium

Carbide Mixtures

Gas released’ BaCO,

added (md 5.0 5.1 4.9 4.8

C,H,

Ba added (mg)

Ratio”

5.1 49 103 252

1:1.02 1:9X 1:21.2 1:52.6

generated (pmol)

Efficiency* (%)

CH,

C&b

‘3-b

H*

1.2 2.9 7.7 9.2

9.2 22.4 62.2 75.5

0.03 0.07 0.07 0.07

ND 0.11 0.2 0.4

ND 0.03 0.08 0.17

33 357 724 1870

Note. ND, not detected. a Variable amounts of barium metal were added to approximately 5 mg barium carbonate to obtain this ratio. * The Ba/BaC, mixtures were hydrolyzed with 2 ml water in evacuated and stoppered 160-ml vials. After the hydrolysis reaction had reached completion (2 h), the resulting gas phase was analyzed by gas chromatography and the % efficiency of C,H, generation was determined on the basis of the detected acetylene and the known amount of BaCO, added to the fusion reaction. ’ The micromoles of each indicated gas which were released during the hydrolysis reaction, as determined by gas chromatography.

sents the product of the reaction of unfused barium metal with the added water. With the highest metal/ carbonate ratio tested, this represented a 200-fold molar excess of H, to acetylene (Table 1). Although the smaller scale of our procedure resulted in a less efficient use and conversion of BaCO, to BaC, compared to those achieved by Cox and Warne (l), this loss of efficiency is in part compensated for by our reduction of the molar ratio of H, generated to acetylene and the small volumes of gas involved. Notably, increases in the scale of production of labeled acetylene beyond those described by Cox and Warne (1) have also been reported to result in decreased yields (7) so it may be that the dimensions of the process described by Cox and Warne (1) represent optimized conditions. From the results shown in Table 1 it is also apparent that the hydrolysis of BaC, gives rise to various products, other than hydrogen, which contaminate the resulting acetylene. These contaminants included methane, ethane, and ethylene which occurred at levels of up to 6% of the total detected hydrocarbon products. In general, the concentration of hydrocarbon contaminants remained proportional to the amount of acetylene gas released. This suggests that contaminant production is also a function of the efficiency of the carbideforming reaction and we have previously described this effect with acetylene generated from calcium carbide (2). A further contaminant that might be expected from the hydrolysis of unreacted BaCO, is CO,. For labeled acetylene generation, the presence of CO, would lead to overestimates of acetylene production if measured by scintillation counting. However, the hydrolysis of BaC, results in a substantial increase in the pH of the reaction mixture to pH > 10. A solution of this high pH minimizes the possibility of acid-catalyzed CO, release from BaCO, and would also act as a suitable trap for any CO, present in the reaction vessel. In confirmation of these possibilities, no increase in CO, concentration was detected by gas chromatography during the hydrolysis

reactions using unlabeled BaC,. We are unable to comment on the effects of our simplifications on the purity of the acetylene generated since those data were not presented by Cox and Warne (1). For our experiments with nitrifying bacteria we required a simple method for making defined and reproducible additions of acetylene to cell suspensions. This was most easily achieved by dissolving acetylene in a solvent. The most applicable solvent for our purposes was DMSO since it is apparently inert in our system, while many other solvents are likely substrates or known inhibitors of ammonia monooxygenase in N. europaea. Because of the high solubility of acetylene in DMSO, over 95% of the acetylene was removed from a typical gas mixture by using four cycles of exposure to DMSO (Fig. 1). Furthermore, the volume of the acetylene was reduced to 2 ml (in the liquid phase) from 14 ml (in the gas phase). The dissolution of acetylene into DMSO also placed the acetylene in a convenient liquid medium which was suitable for storage, dilution, or use in labeling studies. The high solubility of acetylene relative to the other gases generated from the hydrolysis of BaC, also provided another benefit in that the acetylene was significantly purified during the trapping process. After repeated exposure of the gas phase to DMSO, most of the contaminating H, remained in the gas phase and this resulted in a greater than 20-fold purification of acetylene relative to H,. The other hydrocarbon gases generated by the hydrolysis reaction were also less soluble in DMSO than acetylene and this resulted in a net two- to threefold purification of acetylene relative to these gases. When the carbide-generating and DMSO-trapping techniques were applied to acetylene generation from Ba14C0,, we achieved an overall efficiency of 60% for the transfer of 14C label to the DMSO, even without correction for residual acetylene present in the gas phase of the DMSO storage vial. On the basis of the specific activity of the starting Ba14C0, and the selectiv-

352

HYMAN I

-0

1

I

I

2 3 CYCLE NUMBER

4

FIG. 1. Selective removal of acetylene from the gas mixture using DMSO trapping. A gas mixture was produced by adding the appropriate pure gases to a stoppered and evacuated double-chambered 14-ml serum vial which was then filled with air. Acetylene (9 pmol) and hydrogen (440 rmol) were added as representative concentrations obtained from the hydrolysis of Ba/BaC, mixtures (see Table 1). Higher concentrations of methane, ethane, and ethylene than those normally generated by the barium/barium carbide reaction were used (4.1, 0.2, and 3.4 pmol, respectively) so as to accurately determine the effects of DMSO trapping on these minor reaction contaminants. The figure shows the percentage remaining gas phase concentration for (B) Hz, (0) C,H,, (A) CH,, (0) C2H4, and (0) C,H, for each cycle of DMSO trapping, as determined by gas chromatography.

ity of the DMSO-trapping method for acetylene relative to the other C-containing gases, this provides an estimate of the molar concentration of acetylene in the DMSO of approximately 2.6 mM. An overall efficiency of 60% is in agreement with the combined results shown in Table 1 for unlabeled acetylene generation where a ratio of barium to barium carbide of close to 2O:l resulted in slightly greater than 60% efficiency in the conversion of BaCO, to C&H, after hydrolsis. Since the subsequent DMSO trapping of acetylene is approximately 95% efficient for acetylene, and there is little uptake of other C-containing compounds, there is a close agreement between the results obtained with gas chromatographic and liquid scintillation detection methods. This indicates that the method we have described for the generation of acetylene is equally applicable for either labeled or unlabeled starting materials. The final test of the applicability of this simple procedure for [U-14C]acetylene generation is that it fulfills our experimental requirements and provides reproducible results. The fluorogram shown in Fig. 2 illustrates that 15 &i of [U-14C]acetylene obtained by using our present method led to 14C-label incorporation into a polypeptide in N. europaeu of the same apparent M, to that previously characterized by Hyman and Wood (6)

AND

ARP

in an experiment which used 2 mCi of commercially supplied [U-14C]acetylene. Moreover, this result demonstrates that a similar molecular weight polypeptide is also labeled in two other major genera of nitrifying bacteria and that comparable amounts of these polypeptides are present in each species when compared by protein concentration. Together, these results suggest a close relationship between these polypeptides and may reflect catalytic and structural similarities between the monooxygenases from which they are argued to be derived. The development of a simple and effective means of generating radiolabeled acetylene will potentially facilitate the use of this reagent in other biochemical studies of acetylene-sensitive enzymes. However, other applications may have different requirements from those described for our application and it may be necessary to modify the procedure to accommodate individual requirements. For example, different applications may require solvents other than DMSO to trap and store labeled acetylene. Acetylene is soluble in many common solvents (e.g., acetone, ethanol), so suitable alternatives are likely to be available. For other applications, the level of contamination of labeled acetylene may be of significance. Contaminant production in carbide hydrolysis reactions is apparently unavoidable (4). In the current study the small amounts of hydrocarbon contamination did not prevent the incorporation of [U14C]acetylene into ammonia monooxygenase (Fig. 2). On the other hand, the presence of H, in labeled acetylene would be unacceptable for studies of acetylene inhibition of hydrogenase as H, very effectively protects against acetylene inhibition (3). While we have determined the quantities of H, and the major hydrocarbon contaminants in labeled acetylene, it must be determined if these or other contaminants are of concern for

MW

Marker

1

2

3

(W

4s

-

29

-

~

FIG. 2. Covalent incorporation of “C label into similar M, polypeptides in three species of autotrophic nitrifying bacteria. The fluorogram was obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the membrane fractions of three nitrifying bacteria following exposure to [LJ-“C]acetylene generated from Ba’%O,. The bacteria were incubated with [U-“Clacetylene using the incubation conditions described under Materials and Methods. The labeled bands were obtained with (lane 1) N. europaea (apparent M. 26,700), (lane 2) N. oceanua (apparent M. 25,000), and (lane 3) N. multiformis (apparent M, 25,700).

RADIOLABELED

ACETYLENE

each particular application. If contamination of labeled acetylene is of concern, then further purification may be required. We have previously described a simple cryogenic method for generating highly purified acetylene which would be applicable to labeled acetylene (2). ACKNOWLEDGMENT This research was supported Agriculture Grant 88-37120-3956.

by the United

States

Department

of

J. D., and Warne,

2. Hyman, 298-303.

M. R., and Arp,

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1893-1986. Microbial.

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Dalton,

S. W. (1965)

10. Watson, S. W., Graham, (1971) AFC~. Microbial.

Biochemistry

D. J. (1988)

P. M.

R. M., and Sheppard,

8. Prior, S. D., and 1055109.

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H. (1985)

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