Anal. Chem. 1994,66, 4093-4096
Determination of Dimethyl Sulfoxide in Aqueous Solution by an Enzyme-Linked Method A. D. Hatton,tf* G. Malln,'~tA. G. McEwan,*r§ and P. S. Lisst School of Environmental Sciences and School of Biological Sciences, Universiw of East Anglia, Norwich NR4 7TJ, U.K.
A novel and highly specific method has been developed for the determination of dimethyl sulfoxide (DMSO) at nanomolar levels in aqueous solution. After removal of dimethyl sulfide (DMS) from the solution by purging with oxygen-free nitrogen, DMSO is reduced to DMS using the enzyme DMSO reductase purified from the bacterium Rliodobacter capsufatus. The resulting DMS is cryogenically trapped and analyzed by gas chromatography. The detection limit is 0.016 nmol of DMSO per sample (maximum volume 100 cm3), and precision for standards in the concentration range 0.063-1 .O m o l of DMSO is within 2%. The high specificity of the enzymatic reduction step for DMSO was proven in tests with a range of organic sulfur compounds including dimethyl sulfoniopropionate (DMSP, the major biochemical precursor of DMS in algae). The possibility of competitive inhibition of the enzyme was also addressed. In comparison to existing methods for DMSO analysis, the enzymatic method is advantageous in terms of its specificity, its ease of use, and the safe nature of the reducing agent. The technique can be used for freshwater and seawater samples and may also be suitable for pharmaceutical, food, and beverage industry applications. Depth profiles of DMSO, DMS, and DMSP for seawater samples collectedin the Pacific Ocean are presented. Dimethyl sulfoxide (DMSO) is widely used both as an industrial solvent' and for diverse medical applications.24 It occurs naturally in a wide range of beverages and food~tuffs,~Jj in marine and freshwater environment^,^-^ in rainwater,1° and in the atmosphere.l0JI DMSO in seawater arises from photochemical and bacterial oxidation processes and is thought to be a key compound in the marine biogeochemical cycle of dimethyl sulfide (DMS), the most abundant volatile sulfur compound in seawater.I2J3 DMS is biogenically produced in of Environmental Sciences. School of Biological Sciences. 8 Present address: Department of Microbiology, University of Queensland, Brisbane, Queensland 4072, Australia. (1) Ogata, M.; Fujii, T. Ind. Health. 1979, 17, 73-78. (2) Evans, M.S.;Reid,K.H.;Sharp,J.B.,Jr.Neurosci.Lett. 1993,150,145-148. (3) David, N. A. Annu. Rev. Pharmacol. 1972, 12, 343-374. (4) Pommier, R. F.; Woltering, E. A.; Milo, G.; Fletcher, W. S. Anti-Cancer Drugs 1992, 3, 635-639. (5) Pearson, T. W.; Dawson, H. J.; Lackey, H. B. J. Agric. Food Chem. 1981,29, 1091-1093. ( 6 ) de Mora, S. J.; Lee, P.;Shooter, D.; Eschenbruch, R. Am. J . Enol. Vitic. 1993, 44, 327-332. (7) Andreae, M. 0. Limnol. Oceanogr. 1980, 25, 1054-1063. (8) Gibson, J. A. E.; Garrick, R . C.; Burton, H . R.; McTaggart, A. R. Mar. Biol. (Berlin) 1990, 104, 339-346. (9) Ridgeway, R. G.; Thornton, D. C.; Bandy, A. R. J . Atmos. Chem. 1992.14, 53-60. (IO) Harvey, G. R.; Lang, R. F. Geophys. Res. Lett. 1986, 13, 49-51. ( 1 1 ) Berresheim, H.; Tanner, D. J.; Eisele, F. L. Anal. Chem. 1993, 65, 84-86. + School t
0003-2700/94/0366-4093$04.50/0 0 1994 American Chemical Society
surface seawater and enters the atmosphere via sea-air exchange. Once in the atmosphere, it oxidizes to sulfur dioxide, sulfate, and other compounds which can influence the acidity of aerosols and rainwater, particularly in remote regions. Sulfate aerosols derived from DMS also represent a major source of cloud condensation nuclei over the remote oceans and thus influence cloudiness in such regions.14 Current evidence suggests that the quantity of DMS emitted to the atmosphere is only a small portion of the potential marine p00l.l~ Conversion of DMS to other sulfur compounds such as DMSO may represent a significant sink for DMS in the marine sulfur cycle. Likewise, DMSO may be a significant source of DMS following bacterial reduction in some environments. Although DMSO has been detected in seawater, its role in the marine sulfur cycle has yet to be defined since few reliable measurements have been made due to analytical difficulties. Various methods have been reported for DMSO analysis in aqueous samples. Direct measurement of DMSO by gas chromatography is generally insufficiently sensitive for the nanomolar concentration range found in environmental ~ a m p l e s , ~although J ~ J ~ a recently reported method6 achieved a detection limit of 1.25 nmol of DMSO dm-3. Chemical reduction of DMSO to DMS with subsequent analysis of the DMS has greater sensitivity, but existing methods are subject to some interferences. The technique reported by Andreae17 involved the addition of sodium borohydride'or chromium( 11) chloride to reduce DMSO, but this method was found to be unsuitable for samples containing significant levels of dimethyl sulfoniopropionate (DMSP). AnnessI8used acidified stannous chloride to reduce DMSO to DMS in beer samples, and this technique has been used for seawater samples following removal of DMSP by alkali hydroly~is.~,~ Although these methods are adequate for nanomolar DMSO concentrations, specificity is problematic, and the chemical reduction step requires the use of strong acid and alkali solutions. In this paper we report the development of a highly specific enzymelinked method for the detection of DMSO at nanomolar concentrations which overcomes these problems. (12) Kiene, R. P. Microbial sources and sinks for methylated sulfur compounds in the marineenvironment. In Kelly, D. P., Murrell, J. C., Eds.;MicrobialGrowth on C, Compounds, 7; Intercept: London, 1993; pp 15-33. (13) Malin, G.;Turner, S. M.; Liss. P. S. J. Phycol. 1992, 28, 590-597. (14) Charlson, R. J.; Lovelock, J.E.; Andreae, M. 0.;Warren, S. G. Nature 1987, 326, 655-661. (15) Paulin, H. J.; Murphy, J. B.; Larson, R. E. Anal. Chem. 1966, 38,651-652. (16) Wong, K. K.; Mei Wang, G.; Dreyfuss, J.; Schreiber, E. C. J . Inuest. Dermatol. 1971, 56, 44-48. (17) Andreae, M. 0. Anal. Chem. 1980, 52, 15C-153. (18) Anness, B. J. J . Sci. Food Agric. 1981, 32, 353-358.
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994 4093
EXPERIMENTAL SECTION Standards and Reagents. Standards were prepared as follows: 1 cm3of DMSO (99.5% GC grade, Sigma Chemical Co.) was diluted with 0.5 dm3 of distilled water to give a stock solution containing 27.97 mmol of DMSO dm-3. This primary stock solution was then diluted to 0.1 12 pmol of DMSO dm-3 to produce the working standard. A range of volumes between 0.5 and 12 cm3 of this standard, corresponding to 0.056g 1000 1.343 nmol of DMSO, was used for calibration. All standard solutions were stored in volumetric flasks in the dark at 4 OC and were found to be stable over several weeks. It should be I d noted that the distilled water used throughout this study was 01 ,I prepared by reverse osmosis followed by distillation in an all0 10 20 30 Time (min) glass system. Early in the development of the technique, we Flgure 1. Time vs square root of peak area for replicate 10 cm3 experienced erratic background problems when Milli-Q water aliquots of DMSO standard solution, containing 0.625 nmoi of DMSO, was used for reagents and standards. However, we are unable which were purged with oxygen-free nitrogen in the presence of the to state whether these were due to contamination or enzyme reducing solution for different time periods. inhibition. One batch of Analar water (Sigma) was found to be contaminated with 14.6 nmol of DMSO dm-’. drawn into a glass syringe and then expelled, so that the syringe DMSO reductase was purified from cells of Rhodobacter was rinsed and no air remained. The required sample volume cupsulutusstrain H123 using themethodoutlined by McEwan was then taken up and injected through a two-way luer valve et al.19 The purified enzyme appeared as a single band on a into the purge chamber, where it mixed rapidly with the silver stained gel following standard protein gel electrophoresis reducing solution due to bubble-generated turbulence. The (data not shown). Protein determination was by the method mixture was illuminated with three 60 W daylight bulbs. Under of Bradford.2o Consistent results have been obtained with these conditions, EDTA forms radicals which reduce FMN five separately purified batches of DMSO reductase. Flavin mononucleotide (FMN) and ethylenediaminetetraacetic acid to F M N H z , and ~ ~ this acts as an electron donor to DMSO reductase,23 catalyzing the reduction of DMSO to DMS. (EDTA) were obtained from Sigma Chemical Co. Extremes of pH should be avoided as they may affect DMSO A number of compounds were tested for possible interferreductase activity. Since quantitation of the conversion of ence with the enzyme-linked reduction of DMSO to DMS. DMSO to DMS will depend on the rate of DMSO reduction These were dimethyl sulfoniopropionate (DMSP, Research and the efficiency of degassing DMS from the sample, the Plus Inc., Bayonne, NJ), dimethyl sulfone (DMSOz), tritime dependency of the procedure was investigated. Replicate methylamine N-oxide (TMAO), glutathione, methionine, 10 cm3volumes of DMSO standard were purged for between thiamin, cystine, cysteine, biotin, homocysteine, methionine 2 and 30 min, and the reaction was found to reach completion sulfoxide, S-methylmethionine, and S-adenosylmethionine within 15 min (Figure 1). Consequently, a 20 min purge (Sigma Chemical Co.). Stock solutions of each compound period was generally used. However, for larger sample volumes were prepared in distilled water. the purge time was adjusted to account for the purge efficiency, Apparatus. The system for trapping DMS was almost e.g., 100 cm3samples were purged for 30 min. When samples identical to that described by Turner et a1,21with the exception of 50-100 cm3 volume were used, the volume of reducing that the purge tube was illuminated using three 60 W daylight solution added was increased to 5 cm3 to ensure complete bulbs to initiate the FMNH2-dependent reduction of DMSO conversion of DMSO to DMS. It is essential to remove by DMSO reductase. The analytical system consisted of a background levels of DMS prior to the reduction step and Varian 3300 gas chromatograph fitted with a flame photowherever possible to analyze the sample immediately after metric detector and a Chromosil330 column.21 The GC was collection. For field samples we have found it convenient to operated using a temperature program (40-85 “C), and the quantify DMS in a second analytical system and then DMS retention time was 3.4 min. The detector output was immediately analyze this DMS-free sample for DMSO. It monitored on a Hewlett-Packard 3390A reporting integrator. is also possible to add the reducing solution to the purge tube Analytical Procedure. Reduction of DMSO to DMS was acheived using a reducing solution containing 25 Mg ~ m - ~ immediately after DMS analysis. In either case, the purge tube is emptied and rinsed with distilled water between samples. DMSO reductase, 30 mM EDTA, and 540 pM FMN. Different concentrations of the reagents were tested to ensure A typical calibration curve is shown in Figure 2. The yield that they were all present in excess. A 2 cm3 volume of this of the reduction of DMSO to DMS was determined by solution was added to a 50 cm3 purge chamber, which was comparison with thequantity of DMS resulting from the alkali continually bubbled with oxygen-free nitrogen gas (60 cm3 hydrolysis of equivalent concentrations of DMSP, which is min-I). This creates the semianaerobic conditions ideal for known to be 100%efficient.21 Thecalibrationcurves obtained the reduction of DMSO. A small amount of standard was were in very close agreement (Figure 3), suggesting that the overall efficiency of the procedure was 99%. To ensure that
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I
(19) McEwan, A. G.; Ferguson, S. J.; Jackson, J. B. Biochem. J . 1991,274,305307. (20) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (21) Turner, S. M.; Malin, G.; Bagander, L. E.; Leck, C. Mar. Chem. 1990, 29,
47-62.
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Analytical Chemistry, Vol. 66,No. 22, November 15, 1994
I
(22) Massey, V.; Stankovich, M.; Hemmerich, P. Biochemistry 1978, 17, 1-8. (23) McEwan, A. G.; Wetzstein, H. G.; Ferguson, S. J.; Jackson, J . B. Biochim. Biophys. Acfa 1985, 806, 410-417.
,
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Q 2000 4 l i n
Table 2. Naturally Occurring Sulfur Compounds Which Were Tested for Possible Interference with the Enzymlc-Linked DMSO Analysis' compound
dimethyl sulfoxide dimethyl sulfone dimethyl sulfoniopropionate trimethylamine N-oxide glutathione methionine thiamin cystine cysteine homocysteine biotin methionine sulfoxide S-methylmethionine S-adensoylmethionine
c
0
c
e e! 1000
h
I 0
I
I
0,5 1 1.5 Nanomoles DMSO added Figure 2. Calibration curve for DMSO obtained using the enzymelinked reduction method. Standard error values were smaller than the symbols plotted (regression data, y = 39.300 2127.8x, ff = 0.999). 0
+
3000
A solutioncontainingtheequivalent of 1.563nmc. S of eachcompounmwas purged with the reducing solution for 20 min, followed by analysis of the trace gases produced for DMS.
0
5
Sulfur (nmol drnm3) 10 15
5
20
0
30
00 Y
0
I I
I
0
0.5 I 1.5 Nanomoles DMSO or DMSP added Flgure 3. Comparison of standard curves for DMSO (0)and DMSP (0)(regression data for DMSO, y = 28.379 2131,7x, = 0.999; for DMSP, y = 30.025 21 10.lx,ff = 0.997).
+
+
~~
~
~
Table 1. Reproduclbility of DMSO Analyses for Standards In Distilled Water and for a Natural Seawater Sample' sample
standard
mean S D un- 1 CV(%)b
0.067 nmol
0.224 nmol
0.559 nmol
1.006 nmol
seawater 0.11 nmol
212.1 214.1 21 1 205.6 216.7
697.3 707.1 699.4 707.2 700
1876.2 1884.1 1850 1863.1 1854.7
3193.7 3161.8 3152.8 3193.7 3146.4
347.9 331.7 337.6 348.9 359.0
211.9 4.14 1.95
702.2 4.63 0.66
1865.6 14.37 0.77
3169.7 22.6 0.71
345.0 10.6 3.07
*The first five values given are the square roots of the peak areas for replicate analyses. b Coefficient of variation.
the DMSO reduction method was suitable for seawater samples, a calibration curve was prepared using DMSO in artificial seawater, and known additions to natural seawater were also analyzed. In both cases, results were quantitative within the experimental precision. The reproducibility of DMSO analyzes for standards in distilled water and a natural seawater sample is shown in Table 1. The seawater data are for a sample collected in the North Sea just off the East Anglian coast in early March 1994. At this time, relatively low concentrations of DMSO were observed (2.188 nmol dm-3),
d
go
120
I50
012
0.25
0:3 0.35 0.4 Chlorophyll a (ug dm'3)
0.45
0:5
Figure 4. Depth profile for DMS, DMSO, DMSP dlssolved (DMSPd)and particulate (DMSPp) fractions, and chlorophylia, for seawater samples collected in the Pacific Ocean.
and it was necessary to analyze a 50 cm3volume. The enzymelinked DMSO reduction method should be suitable for use with any DMS detection system. Using our GC system, a detection limit of 0.016 nmol of DMSO was obtained, which corresponds to a concentration of 0.156 nmol of DMSO dm-3. The largest sample volume tested was 100 cm3. Interferences. Since the technique involves the use of the enzyme DMSO reductase, we investigated the possibility of competetive inhibition of the enzyme by DMSO2 and TMAO, a known substrate for DMSO reductase.19 Approximately 1.563 nmol of DMSO2 and 1.8 nmol of TMAO were added separately to 0.625 nmol DMSO standards and analyzed for DMSO. Results showed no decrease from the expected concentration. To investigate whether other naturally occurring sulfur compounds produce DMS as a product of the reaction with the reducing solution, 1.563 nmol S aliquots of a number of organic sulfur compounds were analyzed. The results were negative in all cases, indicating that the enzymatic reduction of DMSO to DMS is highly specific (Table 2). Our Analfliml Chemistry, Vol. 66,No. 22, November 15, 1994
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tests included DMSP, a compound which can be present at high concentration in natural s e a ~ a t e r , * ~which . * ~ has been particularly problematic for DMSO detection methods which rely on chemical reduction of DMSO to DMS.*J7-18 Environmental Measurements. The DMSO assay detailed here was tested under field conditions onboard the Columbus Iselin in the equatorial Pacific. A series of depth profiles were analyzed for DMS, DMSO, DMSPparticulate (DMSPp) and dissolved (DMSPd) fractions, and chlorophyll a. In all cases, measurements were performed on the same aliquot of seawater. Samples were stored prior to analysis in ground glass-stopperedbottles with minimal headspace for a maximum of 6 h in the dark at ambient seawater temperature. Figure 4 shows data for a typical profile consisting of nine samples collected between 1 and 150 m depth at 00°30’N, 89OOO’W on Nov. 11,1993. DMSO concentrations ranged from 2.8 13 to 14.375 nmol of DMSO dm-3, with the highest concentration observed near the surface. The variation in DMSO concentration below the surface was similar to that of the other (24) Turner, S.M.; Malin, G.; Liss, P. S.;Harbour, D. S.;Holligan, P. M.Limnol. Oceanogr. 1988, 33, 364-315. ( 2 5 ) Malin, G.;Turner, S.M.; Liss, P. S.;Holligan, P. M.; Harbour, D. S . DeepSea Res. 1993, 40, 1487-1508.
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sulfur compounds, particularly DMSPp. A full analysis of the complete data set will appear elsewhere. We believe that the enzyme-linked DMSO reduction method will prove to be a great asset for future studies of the role of DMSO in the biogeochemical sulfur cycle. It may also be of use for measuring DMSO concentration in food, wines, beers, and samples taken to assess DMSO metabolism following uptake of pharmaceuticals containing DMSO.
ACKNOWLEDGMENT This research was supported by the U.K. Natural Environment Research Council and the University of East Anglia Research Promotion Fund. We would also like to thank Neil Benson for advice on enzyme purification, Sue Turner for advice on DMS analysis and help with analysis of field samples, and the officers, crew, and scientists onboard the Columbus Zselin, Nov. 5-Dec. 1, 1993. Received for review April 8, 1994. Accepted July 30, 1994.’ *Abstract published in Aduance ACS Abstracts. September 15, 1994.
