As halogen sources KBr, NaC1 or a sea salt solution were utilized. The haloform reaction was used as a subsequent reaction to yield the halogenated methanes.
Fresenius' Journal of
Fresenius J Anal Chem (1992) 342:827-833
Formation of C 1/C2-bromo-/chloro-hydrocarbons by haloperoxidase reactions
@ Springer-Verlag1992
B. Walter and K. Ballschmiter
Department of Analytical and Environmental Chemistry, University of Ulna, Albert-Einstein-Allee 11, W-7900 Ulm, Federal Republic of Germany Received August 24, 1991 Summary. We report here the first results of a study of the enzymatic formation of halogenated C1/C2-hydrocarbons from natural, biochemically relevant molecules using enzymes such as chloroperoxidase (CPO) or horseradish peroxidase (HRP). As halogen sources KBr, NaC1 or a sea salt solution were utilized. The haloform reaction was used as a subsequent reaction to yield the halogenated methanes and ethanes from the halogenated substrate molecules. The products of the incubations were analyzed by HRGC/ECD. With these in vitro reactions we attempted to investigate possible biochemical pathways for the formation of some volatile halogenated organic compounds which are assumed to be of biogenic origin. Most of the incubations with KBr formed bromoform as the main product together with dibromo-chloro-methane and 1,1,2,2-tetrachloroethane as by-products. Incubations with NaC1 result in chloroform as the main product in analogy to the formation ofbromoform. The reaction with sea salt yields no major product but a spectrum of halogenated Ct/Cz-hydrocarbons. Incubations of a water extract of algae meal without any further source of C1-/Br--ions yield CHC13 as the main product and other mixed halogenated methanes as side products. Blank reactions were carried out without enzyme, H202 or substrate to show that the products of the incubations are formed enzymatically and to exclude the possibility of normal hypohalogenic reactions of the halide, H202 and the substrate.
1 Introduction
The occurrence of natural organohalogen and specifically organochlorine compounds has found a renewed interest. Their environmental fate can help to assess the impact of the anthropogenic portion of this group of compounds found in the environment. Halogenated methanes (CH3C1, CH3Br, CH3I, CH2C12, CH2Br2, CH212, CH2C1Br, CH2CII, CHC13, CHBr3, CHBrC12, CHBrzC1) [1-8] and the halogenated methyl-phenyl-ethers (anisoles, XzC6Hs-~-(OCH3), (X = Br, C1) [9, 10] can be found worldwide in the marine environment. For the occurrence of these compounds, biogenic as well as typical anthropogenic sources have to be taken into consideration [11-13]. Three pathways for the nonanthropogenic formation of organochlorine can be discussed:
Offprint requests to: K. Ballschmiter
1) Exchange of iodine/bromine by chlorine in compounds that are primarily of biogenic origin either by a hv-driven radical mechanism or by a Br-/C1-- or an I-/C1--exchange, e.g. [6]: CH3I/CH3Br -~ CH3C1 CH212/CH2Br 2 - . CH2C12 2) Initial formation of the C - Hal-bond by the reaction of an activated C - H - b o n d , [e.g. in the c~-position t o a carbonyl( > C = O) or carboxyl-group ( - C O O H ) ] with haloperoxidases followed by hydrolysis of the (Hal)xC- (C = O)-bond [14, 15]: C 1 H z C - C O O H + HzO ~ CH3C1 + H2CO3 3) The direct halogenation of the aromatic system of the anisols or phenols, e.g. [16, 17]. Haloperoxidases are enzymes which are widely distributed throughout nature [18]. They are able to perform a multitude of reactions, especially halogenation reactions, following the general equation [19]: substrate + H202 + X - + H + e,zyme , halogenatedproduct + 2 H 2 0 In this reaction the formation of HOX is considered to be the relevant intermediate step of the halogenation [20], but another mechanism with an enzyme-bound intermediate, depending on the presence of a substrate and the type of halide-ion is also discussed [18, 19, 21]. Haloperoxidases, especially bromoperoxidases, can be found in marine macroalgae as well as in horseradish or milk [18]. Macroalgae are also the source of a multitude ofhalogenated compounds [18, 22-25]. Chloroperoxidase (CPO), first isolated by L. P. Hager et al. [26-28], has been found in Caldariomyces fumago. The enzyme has a mol weight of about 40,000 and contains 2 5 - 3 0 % carbohydrates using ferriprotoporphyrin IX as the prosthetic group. Chhw~phenols, e.g. have been detected in fungi [18, 29]. The fungal chloro-metabolites have the chlorine atoms mainly bonded to a five or six membered ring [29], sometimes the dichloroacetic acid is part of the metabolite. A 4-methyl-tetrachlorophenol (Drosophilin A) has been isolated from the insect Drosophila substrata [30]. Various lichens contain chlorinated and hydroxylated xanthones [31]. The biosynthesis of many of the chloro-metabolites has been extensively reviewed by Turner [32]. To evaluate the possible formation of volatile halogenated C1/C2-hydrocarbons several in vitro incubations with chloroperoxidase (CPO) and horseradish
828 peroxidase (HRP) were carried out with substrate molecules that are common in biochemistry and which possess a carbonyl-activated site (acetic acid, acetone or pyruvic acid). KBr (Suprapur), NaC1 (Suprapur), or an artificial sea salt mixture were used as the halogen source. To obtain the halogenated methanes and ethanes from the halogenated substrates, the HalxC - (C = O)-bond was cleaved at a pH of 7 - 1 0 following the haloform reaction mechanism. Incubations without an alkaline hydrolysis of the primary formed products (halogenated substrates) were also carried out [33]. Blank reactions without an enzyme, without a substrate or without H2Oz but under the same conditions as the enzyme incubations were carried out to ascertain that the products have been formed by enzymatic reactions and not by the spontaneous reaction of the halide-ion X-/HzO zsystem. In a series of experiments the H202 used by the chloroperoxidase was generated by a glucose oxidase system. No external H2O 2 was added and only the dissolved oxygen was used by the glucose oxidase. Oxidases are of the twoelectron transfer type and react with oxygen as acceptor for hydrogen to form H202 [34]. Amino acid oxidases and xanthin oxidases belong to the same group as glucose oxidase. The oxidases lead to e-ketoacids, thus producing the I-t202 and possibly preferred substrates for the haloperoxidases.
2 Experimental 2.1 Incubation
The following specification lists the general composition and the approximate concentration ranges of the enzyme incubation mixtures: 2.1.1 H202, halogen-ion, haloperoxidase 1) 20 ml of 25 - 100 mmol/1 phosphate buffer solution, pH 3 or 6; 2) I ml of 2 tool/1 KBr-solution (Suprapur, Merck) [2 mmol B r - ] or 10 ml of 0.1 tool/1NaCl-solution (Suprapur, Merck [1 mmol C1-] or 10 ml of the artificial sea salt solution [100g/i]; 3) l - 2 m m o l substrate; 4) 100gl Perhydrol (Merck), solution of 30% H202 [0.9 mmol]; 5) 2 0 - 2 0 0 gl haloperoxidase [ 1 0 0 - 2000 units]; 5.1) chloroperoxidase (CPO) or 5.2) horseradish peroxidase (HRP). 2.1.2 Glucose oxidase system, halogen-ion, haloperoxidase 1) 20 ml of 25 - 100 retool/1 phosphate buffer solution, pH 3 or 6; 2) I ml of 2 tool/1 KBr-solution (Suprapur, Merck) [2 mmol Br-]; 3) 1.8 mmol glucose; 4) 1 ml glucose oxidase [1100 units]; 5) 2 0 - 2 0 0 gl haloperoxidase (as above). The incubations were started by adding the enzyme to the buffer-substrate mixture and stirring the mixture in a 50 ml-flask with a magnetic stirrer at room temperature ( 2 3 - 2 5 ° C). After a given time, e.g. 1 h, the mixture was either directly extracted with I ml hexane (Nanograde or EOC1, Promochem) or extracted after adjusting the pH to 7 - 1 0 with 0.5 mol/1 K2CO3-solution. The separation of the two phases was carried out using a small Weil-Quentinadapter and a NazSO4-microcolumn was used to dry the hexane phase. An aliquot of this extract (about 2 gl) was
analyzed by H R G C / E C D . Identification was performed by comparison of the retention times with reference compounds. 2.2 Instrumentation
Gas chromatograph: Hewlett Packard 5890 with ECD, injector: splitless/split 2min, 200°C, detector 300~'C. Stationary phases: a) HP 1 (Hewlett Packard), 10 m, 0.53 m m i.d., film thickness 2.65 gin. b) DB 624 WCOT (J & W Scientific), 30 m. 0.53 m m i.d., film thickness 3 gin. Carrier gas: Nitrogen 5.0 (Linde), velocity: a) 20cm/s, b) 14 cm/s. Make-up gas: Ar/CH4 (90/10), flow 40 ml/min. Temperature programs: a) 40°C, 2 min isothermal, 40 to 120°C with 5°C/min, 120 to 250°C with 15°C/min, 5 min isothermal or b) 40 ° C, 2 rain isothermal, 40 to 200°C with 4°C/rain, 200 to 250°C with 10°C/rain, 10 rain isothermal. Computing integrator: Shimadzu C-R4A Chromatopac.
3 Results and disenssion 3.1 Blank reactions
Different blank reactions have been studied. 3.1.1 Blank reactions without enzyme The blank reactions without haloperoxidase were carried out under the same conditions as the enzyme incubations. All these blank reactions showed no formation of halogenated Ca/C2-hydrocarbons. 3.1.2 Blank reactions without
H202
The chromatogram of an incubation without hydrogenperoxide but under the same conditions as with H2Oz (substrate, KBr and CPO in a phosphate buffer) showed no peaks for volatile halogenated hydrocarbons. This means that the haloperoxidase CPO is really a peroxidase which needs H202 for an enzymatic reaction (halogenation) and cannot use Oz from the air to carry out enzymatic reactions. Thus it is not necessary to work in an inert atmosphere or with the exclusion of air from the reaction flask. Since H202 is a toxin for living cells and there are a number of enzymes which are able to destroy hydrogen peroxide (e. g. catalase), CPO could also have a protective effect and destroy H202 in the cell in order to protect the organism. The halogenation reaction would be a following step in the presence of a halide and an organic substrate. AH2 + enzyme ~ A + enzymeHz enzymeH2 + 02 ~ enzyme + H 2 0 2 Peroxidases: a) AH2 + H202 ~ A + 2 H20 b) HC1 + H202 ~ HOC1 + H 2 0 HOC1 + R H ~ R - C1 + H 2 0 • Katalase." 2 H202 -~ 2 H 2 0 + O z . Oxidases:
.
3.1.3 Blank reaction without a substrate The incubations with CPO, KBr/NaC1 and H2Oz in a phosphate buffer without a specific substrate yielded CHBrzC1, CHBr3 and probably C H C l z - CHClz in various concentrations, depending on the amount of CPO, halide or
829 Table 1. Reaction products of the blank reactions with CPO and without a substrate Composition of the incubation mixture
pH
Reaction products
480 units CPO, 2 mmol KBr, 0.9 mmol HaO 2
3.1 ~ 7.0
CHBr3 (170 ng/incubation), CHBr2C1
480 units CPO, 2 mmol KBr, 0.9 mmol H202
3.0 --*7.2
CHBr3 (160 ng/incubation), CHBr2C1
480 units CPO, 2 mmol KBr, 0.2 mmol H202
3.3 -~ 7.3
CHBr3 (230 ng/incubation), CHBrEC1
480 units CPO, 20 gmol KBr, 0.9 mmol H202
3.5 ~ 7.2
CHBra (310 ng/incubation), CHBr2C1
480 units CPO, 20 gmol KBr, 90 gmol H2Oz
3.2 ~ 7.2
CHBra (30 ng/incubation), CHBr2C1
600 units CPO, 2 mmol KBr, 0.9 mmol H202
3.2 - . 7.2
CHBra (160 ng/incubation), CHBr2C1, CHC12- CHC12
150 units CPO, 40 gmol KBr, 4509 gmol HzO2
3.2-~ 7.2
CHBr3 (30 ng/incubation), CHC12- CHC12
150 units CPO, 20 gmol NaC1, 450 p.mol H2Oz
3.1 --*7.2
Small amounts of CHBr2C1 and CHBrC12
Table 2. Reaction products of incubations of chloroperoxidase, KBr, H202 with several substrates. The arrow (-~) represents an alkaline hydrolysis Substrate
pH
Reaction products
Acetic acid (0.9-3.4 mmol)
3 ~ |0
CHBr3" (130, 170, 260, 270, 370 ng), CH2Br2, CHBr2Ct, CHBrC12, CHEBr- CH2Br, CHC12 - CHC12, CC12 = CC12
Acetic acid (1.8 mmol)
6
CHBr3" (100 ng), CH2Br-CH2Br, C H C I 2 - CHCI2
Propionic acid (1.3 mmol)
3 ~ 10
CHBra (420 ng), CHBr2C1, CHC12-CHC1/
Propionic acid (1.3 mmol)
3
CHBr3" (90 ng), CHBrzC1, CHC12-CHC12
2-Keto-propionic acid (Pyruvic acid) (1.4 mmol)
3 ~ 10
CHzBr2, CHBrzC1, C H / B r - C H 2 B r
Butan-dicarbonic acid (Succinic acid) (2.0 mmol)
3 ~ 10
CHBra a (300 ng), CHzBr2, CHBr2C1, CCI2 = CC12
Butan-dicarbonic acid (Succinic acid) (2.0 mmol)
3
CHBra" (I 00 ng), CH2Br2, CHBrzCI, CC12 = CC12
Acetylacetone (2.3- 3.5 mmol)
3 ~ 10
CHzBr2, CHBrEC1, CHzBr-CH2Br, CCI2 = CCI2
Acetone (2.7 mmol) (480/950 u CPO)
3 -~ 10
CHBr3 (680/1100 ng), CHzBr2, CHBrzC1, CHBrC12, CH2Br- CHzBr, CHCI2 - CHC12, CCIE = CCl2
Citric acid (2.0 mmol)
3 -~ 7
CHBra (2300 ng), CHBrzC1, CHClz-CHC12
Citric acid (2.0 mmol)
3
CHBr3 (780 ng), CHBrzC1, CHzBr-CHzBr, CHClz-CHClz
Citric acid (2.0 mmol) 3 The KBr-concentration was 10 times lower in this incubation as above.
CHBr3 (660 ng), CHBrzC1, CHCI2-CHCI2
D(+)-glucose (1.8 mmol)
3~ 9
CHBr3 a (300 ng), CC14, CHBr2C1, CHCI2- CHCI2
D(+)-glucose (1.8 mmol)
3
CHBra" (90 ng), CHC13, CH2C1-CCI3, CHzBr-CH2Br
D(+)-glucose (1.8 mmol)
6
CHBra a (15 ng), CHBrzC1, CHC12- CHC12
a The concentration of the CHBr3 is in the same range as the CHBr3 formation in a blank reaction without a substrate (see 3.1.3). The concentration of CHBr3 is given in ng/incubation
H202, used in the incubation mixture. F u r t h e r investigations are in progress to confirm the identification of 1,1,2,2tetrachloroethane, because it could not be separated from l , l - d i b r o m o a c e t o n e with these stationary phases under the conditions described above. The results are shown in Table 1. Several explanations can be given for the occurrence of this p r o d u c t pattern, which is very similar to those from some substrate incubations: CPO itself m a y serve as a substrate for a h a l o g e n a t i o n reaction at which the glyco-protein b o d y o f the enzyme ( M W : >40,000) acts as the target site for the reaction with H O X . This glyco-protein-residue will have enough activated sites which can be used as substrates. The
enzyme-suspension, which consists o f the enzyme and a sodium p h o s p h a t e solution at p H 4, could also be the source of these compounds. We cannot exclude the possibility that a c o n t a m i n a t i o n of this suspension with halogenated organic molecules has occurred. On the other h a n d it is likely that the enzyme suspension was c o n t a m i n a t e d with traces of organic molecules arising from the p r o d u c t i o n process, which could also serve as substrates for a halogenation reaction. Thus, the results reported in Table 2 need to be corrected using the d a t a obtained in these b l a n k reactions, assuming a "blank f o r m a t i o n " of the referring c o m p o u n d s occurs in an incubation with a substrate, too.
830
U
CHC13
/
\
CI2-CHCI
CHBi Iz
U
ol
CCI2=CCI2 CH2B~-CH~3r I
I
0
4
8
I
I
"
12
16
T(MIN)
I"
Fig. 1. HRGC(HP t)/ECD-chromatogram of an incubation of acetone with CPO, H2Oz and KBr. The identification of CHC12CHCI2 remains to be verified with a different stationary phase J
3.2 Incubations with CPO, KBr and several substrates Table 2 lists the reaction products of the incubations of chloroperoxidase and KBr with a variety of substrates. The CHBr3 formed was quantified by an external quantitation and the concentration is given in parenthesis in ng/incubation. If an alkaline hydrolysis was carried out this is shown by an arrow (--,) at the pH-value. Figure 1 shows a typical gas chromatogram of the reaction products from an incubation with acetone, KBr, H202 and CPO. The compounds marked with U have not been identified yet. Summarizing the results of these incubations, the following conclusions can be drawn. a) The formation of chlorinated compounds as well as brominated products is remarkable but has already been observed in other enzymatic incubations [16, 33]. The source for the chlorine is the C1--contamination of the KBr Suprapur used (max. 500 ppm C1-) and the other reagents that are present in the reaction mixture. The chlorinated compounds could also be formed by Br/C1 exchange reactions as mentioned previously. The extent to which these
rCl2 CHBrzCI
CHC12-CHC12
I
[
I
I
I
0
4
8
].2
16
t(MIN)
Fig. 2. HRGC(HP t)/ECD-chromatogram of an incubation of acetone with CPO, H a O 2 and NaC1
exchange reactions can occur under the conditions of the enzyme reactions is still open. b) The main product of all the incubations with CPO/ KBr, except those with acetylacetone and pyruvic acid, was bromoform. It is not understood yet why these two substrates do not form bromoform. c) When alkaline hydrolysis is carried out after the halogenation step the yields of bromoform are 2 to 3 times higher than without such alkaline hydrolysis. As the spontaneous hydrolysis of the bromoacetones at pH 3 or 6 is rather slow, it could mean that the chloroperoxidase possesses the ability to cleave the C - C bond between the
831 halogenated C-atom and the carbonyl-group and to form directly halogenated methanes. d) The product pattern of the C1/C2-halocarbons of all the incubations, with the exception of those mentioned in b), is quite similar. That means that the product formation only depends on the substrate to a small extent. e) When comparing the different incubations with citric acid it can be seen that the formation of CHBr3 does not depend on the KBr-concentration, at least in this concentration range. f) The influence of the concentration of the CPO on the yield of bromoform is considerable as can be seen in the incubations with acetone. g) The formation of the unsaturated compounds CHC1 = CC1 and CC12 = CC12 can be explained by a hydrolysis of the corresponding precursor molecules C H C I 2 CHC12 and CHC12-CC13. The reaction mechanism of the formation of the haloethanes ( C H 2 B r - C H 2 B r , C H C l z CHC12, CHCI2-CC13) is still open. It could involve the reaction of two CHxXy-radicals or, more likely, a concerted reaction of two HCC12 - C(O)R-molecules as follows:
C ,[Br2CI
Z:
,3
C1-CCI 2 3HB~C1a
.) i
CHCI2-CHCI2A
/ t
/
2 C 1 2 H C - C O O H -~ C H C I 2 - C H C 1 2 + (COOH)2. In the case of haloacetones the cyclopropanone intermediate of the Favorskii rearrangement would undergo a specific two step haloform-type hydrolysis: C1 C1 cIC-C(O)-CCl Br Br 2 H20
(on-) ,
-Br+/-Br-
CHCI2-CHC12
C(O) /\ C1C-CC1 C1 C1
CCI2=CCI z
I
I
I
I
I
0
4
8
12
16
T(MIN)
Fig. 3. HRGC(HP 1)/ECD-chromatogram of an incubation of acetone with CPO, H202 and sea salt
+ H2C03
This mechanism would explain the formation of the higher chlorinated ethanes. Increasing the acidity of the e-hydrogen eases the initial step of the cyclopropanoneformation. This is the removal of a proton to form an enolate ion which stabilizes itself by the ejection of a halide ion [35]. After the Favorskii rearrangement, normal hydrolytic C - C-bond cleavage is possible. Hydrolytic cleavage of the cyclopropanone-intermediate would lead to formation of propionic acid from acetone. The occurrence of CHClzBr and CHCI2 - CHC12 could be interconnected by a preferred leaving of both Br + and Br-. Br + would form the HOBr moiety. 3.3 Incubations with CPO and NaCl
The main product of the incubation with acetone, NaC1 and CPO is chloroform in analogy to the generation of bromoform with KBr. CHCI=CC12, CC12=CC12, CHBrCI2, CHBrzC1, C H 2 B r - C H z B r and small amounts of CHBr3 and C H C 1 2 - C H C I 2 (brominated hydrocarbons probably from the Br--contamination of the NaC1 used) can be identified as side products. Figure 2 shows the reaction products in the H R G C ( H P 1)/ECD-chromatogram of this incubation. Direct halogenation of the substrates with NaOC1 does not lead to the same product pattern. The reaction of acetone with NaOC1 leads to CHC13 and CHzC1-CC13 as main products and to 1,1-dichloroacetone and CC14 as side products. Compared with the pattern of an incubation of acetone with CPO/H202/C1- contains only chloroform as the same
main product. The difficulties of this comparison are the concentrations of the reactive species in these reaction mixtures. The formation of NaOC1 in a CPO-incubation cannot be predicted, therefore the concentration of NaOC1 in a direct halogenation reaction is not necessarily the same as in the enzyme reaction. 3.4 Incubation with CPO and sea salt
The halide-concentrations in this mixture can be assumed to be in the same range as in real sea water as it is used as saline aquarium water. The mixture was diluted so that real sea water concentrations were obtained as suggested by the supplier. The halide concentrations of real sea water are [36]: C1- : 19000 mg/1, B r - : 65 mg/1, I - , IO3- : 0.060 mg/1. The incubation with acetone, CPO and the artificial sea salt mixture yields the following products: CHBrC12, CHBrzC1, CHBr3, CHCI=CCI2, CC12=CC12, C H z B r CH2Br and CHC12-CHCI2. The reaction products can be seen in Fig. 3. 3.5 Incubation with horseradish peroxidase ( H R P ) and KBr
3.5.1 Acetone as substrate The chromatogram gives one major peak (Rt = 25.292 rain; Rt(CHBr3) would be 29.920 rain) which has not yet been identified. Besides this peak, CHBr2C1, CHBr3, C H C 1 2 CHC12 and CC12 = CCl2 can be detected. Since H R P is only able to brominate organic substrates [18], a halogen exchange is likely for the occurrence of the chlorinated compounds.
832 Table 3. Reaction products of incubations with a water extract of milled algae, H202 and CPO Composition of the incubation mixture
pH
Reaction products
20 ml AEH, 0.9 mmol HzO2, 480 units CPO
4 ~ 7.3
blank level
20 ml AEH, 9 mmol HzO2, 480 units CPO
4 --+7.3
CHC13 (+), CHBrC12 (+)
10 ml AEH, 9 mmol H202 , 480 units CPO
3.7 --, 7.2
CHC13 (+ +), CHBrClz (+ +), CHBrzCI (+)
1 ml AEH, 9 mmol H202, 480 units CPO
3.1 ~ 7.2
CHC13 (+ + +), CHBrC12 (+ + +), CHBrzC1 (+)
Blank AEH, 9 mmol H 2 0 2
3.1 ~ 7.2
blank level
1 ml AEC 1, 9 mmol H 2 0 2 , 480 units CPO
3.1 ~7.2
CHC13 (+ + +), CHBrC12 (+ + +), CHBrzC1 (+)
t0 ml AEC 1, 9 mmol HzO2, 2000 units HRP (suspension)
5.7 ~ 7.2
CHBr3 (+)
10 ml AEC 1, 9 mmol H202, 2500 units HRP (crystalline)
5.8 ~ 7.2
CHBr3 (+)
25 ml AEC 2, 18 mmol H202, no CPO nor HRP
5.5 --*7.2
CHBrClz (+); CHzBr-CHzBr (+)
(+), (+ +), (+ + +) indicate the amount of the compounds formed for a rough inter-comparison of the incubations AEH: Algae Extract Hot AEC: Algae Extract Cold
3.5.2 D(+)-glucose and citric acid as substrates Surprisingly, only very small amounts of chlorinated products can be identified in the chromatograms of these incubations: CHC12 - CHC12 and CHC1 = CHCI2.
3.6 Incubations with CPO, KBr, glucose and glucose oxidase In these incubations the hydrogen peroxide which is necessary for the halogenation reaction is formed in situ from glucose and the O2 dissolved in the reaction mixture with the help of the enzyme glucose oxidase. Oxidation of the glucose by the oxidase leads to H 2 0 2 . The glucose serves at the same time as the substrate for the halogenation by the chloroperoxidase and as reducing agent for the H2Oz-production. The maximum of the activity of glucose oxidase is at pH 7, which is quite different from the activity maximum of CPO (pH 3). 3.6.1 Incubation at pH 6 without alkaline hydrolysis Small amounts of CHBrzC1, CHBr3 and probably CHC12 CHClz can be detected. 3.6.2 Incubation at pH 3 without alkaline hydrolysis Compared with the incubation at pH 6.0 only very small amounts of CHBr3 and CHC12-CHCI2 can be detected; the assumed CHC12-CHC12-peak is now greater than the CHBr3-peak. 3.6.3 Incubation at pH 3 followed by alkaline hydrolysis Only very small traces of CHBr3 and CHCI2-CHC12 can be found. The CHBr3-peak is a factor 5 higher than in 3.6.2, and the CHClz-CHC12-peak is the same height. The results show that the production of H202 at pH 6 is high enough for an enzymatic halogenation to occur, even though the activity maximum of CPO lies at pH 3. At pH 3.0 the activity of glucose oxidase and with it the H202-production is too low for a significant halogenation reaction; a
subsequent alkaline hydrolysis does not increase the yield. This means that the limiting step for the production of the halogenated Ct/Cz-hydrocarbons is the halogenation of the substrate and not the cleavage of the C - C - b o n d of a halogenated precursor.
3.7 Incubations with water extracts of dried algae In a series of incubations, the water extract of dried algae was used both as the substrate pool as well as the halogen source; only H 2 0 2 and CPO were added. The dried and milled algae were purchased as a crude powder ("Algenmehl - Meerwunder") supplied for use as a fertilizer. 30 g of this dried algae were extracted with 300 ml of water [bidistilled and extracted with hexane EOC1 (Promochem)] in a Soxhlet-apparatus for 24 h. This extract is named AEH (Algae Extract Hot). Another 30 g of the dried algae meal was extracted at room temperature with 300 ml of water in a 500 ml flask. After 96 h an aliquot of 65 ml was taken from this extract, filtered and named AEC 1 (Algae Extract Cold 1). The rest of this extract was kept in the flask and named AEC 2. Different volumes of the algae extracts, H202 and haloperoxidase were added to 20 ml of either a phosphate buffer pH 3 or pH 6. Table 3 details the composition and gives the results of the incubations. After the halogenation the pH of the incubation mixture was controlled with a pHelectrode and adjusted to 7 . 2 - 7 . 3 with a 0.5 tool/1 KzCO3solution to perform the cleavage of the C - C - b o n d and to form the halogenated methanes and ethanes. The formation of the halogenated methanes shows that a water extract of dried algae contains enough substrates and halides to form mainly chloroform and other mixed halogenated C~hydrocarbons. The quantity of H202 that is necessary to yield halogenated compounds is remarkable high and it is likely that the extract contains many oxidizable molecules that consume the hydrogen peroxide. Since these oxidations may be kinetically and thermodynamically favoured a certain basic level of H 2 0 2 is used for these oxidation reactions. Only when this HzO2-1evel is exceeded an enzymatic halogenation can take place.
833 The product pattern throughout all the CPO-incubations is quite similar. CHC13 is the main product, CHBrC12 and CHBr2C1 occur as side products. In one incubation, traces of CC14 could be detected, but this may be due to contamination. Increased H202 concentration and reduced algae content both lead to increase in the yield of all the compounds. Thus, the effective H202-concentration that can be used for an enzymatic halogenation becomes the limiting factor in the formation of the halogenated components. With horseradish peroxidase (HRP), only b r o m o f o r m is formed (in small amounts), whereas in CPOincubations only chlorinated and mixed halogenated compounds are formed but no CHBr3. The results reflect the ability of the haloperoxidases to form either chlorinated and brominated or only brominated compounds. If crude, unfiltered cold extract o f the algae meal (AEC 2) is incubated without an enzyme, halogenated products can be detected in small concentrations. The occurrence of these compounds is unexpected. In the cold algae extract (AEC 2) a halogenation potential is available that is missing in the Soxhlet-extract because the proteins of the enzymes are destroyed at the high temperatures of the boiling water. It is also interesting that 1,2-dibromoethane can be detected only in this incubation. Also the absence o f bromoform needs to be explained because this is a kind of indicator molecule for enzymatic bromination and occurs in almost all other B r - incubations. The blank reaction of the hot algae (AEH) with H202 but without CPO shows no formation of halogenated C1/C2-hydrocarbons.
4 Conclusion The formation of a broad spectrum o f halogenated C1/C2hydrocarbons by the enzymatic halogenation o f biochemically relevant molecules has been shown to be possible. Further experiments need to be carried out to ascertain to what extent the results can be transferred to in vivo processes and to solve the separation- and identification-problems of some compounds. The ability of the enzymes to use different halides for the halogenation needs to be tested and has to be taken into account when chloro- and bromochlorocompounds are found in coastal areas. It is reported that most of the algae contain mainly bromoperoxidases. The cleavage of the C - C - b o n d by alkaline hydrolysis after halogenation has taken place, cannot be presumed to occur in biochemical pathways in an organism, though it is has been suggested that some algae possess areas (e. g. on their surface) with a p H which is quite different from the normal physiological pH-value [37]. In this context it is important to keep in mind that a number of different haloperoxidases can function in a pH-range varying from acid over neutral to slightly alkaline.
References
1. Lovelock JE, Maggs RJ, Wade RJ (1973) Nature 241 : 194-- 196 2. Singh HB, Salas LJ, Shigaishi H, Scribuer E (1979) Science 203:899-903
3. Rasmussen RA, Rasmussen LE, Khalil MAK, Dalluge RW (1980) J Geophys Res 85:7350-7356 4. Khalil MAK, Rasmussen RA, Hoyt SD (1983) Tellus 35B : 266- 274 5. Class TJ, Kohnle R, Ballschmiter K (1986) Chemosphere 15:429--436 6. Class TJ, Ballschmiter K (1987) Fresenius Z Anal Chem 327:40- 41 7. Class TJ, Ballschmiter K (1988) J Atmos Chem 6 : 3 5 - 4 6 8. Wiedmann T (1989) Diplomarbeit, University of Ulm, Ulm 9. Atlas E, Sullivan K, Giam CS (1986) Atmos Environ 20:12171220 10. Wittlinger R, Ballschmiter K (1990) Fresenius J Anal Chem 336:193-200 11. Gschwend PM, MacFarlane JK (1986) Polybromomethanes. In: Sohn ML (ed) Organic marine geochemistry, ACS Symposium series 305, Florida 12. Manley SL, Dastoor MN (1987) Limnol Oceanogr 32:709715 13. Wever R, Tromp MGM, Krenn BE, Marjani A, Van Tol M (1991) Environ Sci Technol 25 : 446- 449 14. Moore RE (1977) Acc Chem Res 10:40-47 15. Beissner RS, Guilford WJ, Coates RM, Hager LP (1981) Biochemistry 20 : 3724- 3731 16. Walter B (1989) Diplomarbeit, University ofUlm, Ulm 17. Wannstedt C, Rotella D, Siuda JF (1990) Bull Environ Contam Toxicol 44: 282- 287 18. Neidleman SL, Geigert J (1986) Biohalogenations: principles, basic roles and applications. Ellis Horwood, Chichester, UK 19. Hager LP, Hollenberg PF, Rand-Melt T, Chiang R, Doubek D (1975) Ann NY Acad Sci 244:80-93 20. Brown FS, Hager LP (1967) J Am Chem Soc 89:719-720 21. Dunford LIB, Lambeir AM, Kashem MA, Pickard M (1987) Arch Biochem Biophys 252:292-302 22. Fenical.W (1975) J Phycol 11:245-259 23. Faulkner DJ (1982) Natural organohalogen compounds. In: Hutzinger O (ed) The handbook of environmental chemistry, vol 1 part A. Springer, Berlin Heidelberg New York, pp 229254 24. Fenical W (1982) Science 215:923-928 25. Faulkner DJ (1988) Chemistry and application (International Conference on Chemistry and Applications) Bromine and its compounds, 1st, Salford, pp 121-144 26. Beckwith JR, Hager LP (1963) J Biol Chem 238:3091-3094 27. Hager LP, Morris DR, Brown FS, Eberweins H (1966) J Biol Chem 241:1769-1777 28, Morris DR, Hager LP (1966) J Biol Chem 241 : 1763 - 1768 29, Doonan S (1973) The biochemistry of carbon-halogen compounds. In: Patai S (ed) The chemistry of the carbon-halogen bond. Wiley, London, pp 865-916 30. Kavanagh F, Hervey A, Robbins WJ (1952) Proc Natl Acad Sci 38 : 555- 560 31. Santesson J (1969) Ark Kemi 30:449-454 32. Turner WB (1971) Fungal metabolites. Academic Press, London 33. Walter B, Ballschmiter K (1991) Fresenius J Anal Chem 341:564- 566 34. Schlegel HG (1985) Allgemeine Mikrobiologie, 6th edn. Thieme, Stuttgart New York, p 244 35. Loftfield RB (1961) J Am Chem Soc 73:4707-4714 36. Tardent P (1979) Meeresbiologie. Thieme, Stuttgart, pp 186, 188 37. Raven FA, Johnston AM, MacFarlane JJ (1990) Carbon metabolism. In: Cole KM, Sheath RG (eds) Biology of the Red Algae. Cambridge University Press, Cambridge, p 176
