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Metabolic conversion of cyclohexane by Pacific salmon microsomal preparations
Marine Environmental Research 17 (1985) 129-132
Metabolic Conversion of Cyclohexane by Pacific Salmon Microsomal Preparations John M. Kennish, Colleen Montoya, Jane Whitsett & John S. French Department of Chemistry, University of Alaska, Anchorage, Anchorage, Alaska 99508, USA
Hydrocarbons are being introduced into the marine environment from a variety of sources including combustion processes, crude oil spills, fuel oil spills, and controlled disposal such as processed ballast water. Even in the relatively clean waters of Alaska hydrocarbons are being released at an alarming rate. The purpose of this study was to determine the optimum conditions for the metabolism of a representative hydrocarbon by Coho Salmon Oncorhynchus kisutch liver microsomes. The product of cyclohexane metabolism in the salmon microsomal system was identified by gas chromatography-mass spectrometry as cyclohexanol. Conditions of the microsome incubation were varied systematically to determine the optimum temperature, pH, and ionic strength for cyclohexanol production. Cyclohexanol was quantified by capillary column gas chromatography. Maximum cyclohexanol formation was achieved at 20 ° C, a p H of 8.0-8.5 and an ionic strength of 0"026. A linear rate of cyclohexanol formation is seen from 0-60 rain of incubation and there is an apparent decrease in the rate from 60-90 rain. Poor stability of the microsomal preparation from the species studied was also identified and several stability studies have been undertaken using cyclohexane metabolism as a monitor. Adult Coho Salmon were caught by gillnet in Cook Inlet, Alaska. The fish were bled and the livers removed and stored on ice until processed. The livers were pooled and microsomes were prepared according to the method of Coon et al. 1 The microsomes were extracted with 0.25 M sucrose buffer to remove cytosolic factors and the final pellet was resuspended in 0-05 M Tris-C1 buffer (pH 7.5) containing 0.1 .~ KC1, 129 Marine Environ. Res. 0141-1136/85/$03-30 © Elsevier Applied Science Publishers Ltd,
England, 1985. Printed in Great Britain
130
John M. Kennish, Colleen Montoya, Jane Whitsett. John S. French
0-lmst EDTA, 0.01mM phenylmethane-sulfonyl fluoride. 0-01mM dithiothreitol and 20 ° o glycerol. The resuspended microsomes contained 19.1mgml of protein z and a P-450 concentration of 0.630/2M. 3 The microsomes were stored at - 3 0 ° C until used. Unless otherwise indicated, incubation mixtures were prepared in a 2 ml volume consisting of the microsomal preparation, 0.05.~I Tris-Cl buffer (pH 7.5), 0.1M KCI, 1.0mM NADPH, and ..~,'~n°//oglycerol. After addition of 10/21 ofcyclohexane the mixtures were incubated in a shaking water bath at constant temperature. The samples were extracted with 1.0ml of methylene chloride containing the internal standard (lhexanol). The organic phase was removed and concentrated to 50/21 with nitrogen. Analysis of cyclohexanol was accomplished using a Hewlett Packard 5840A gas chromatograph equipped with a flame ionization detector and a WCOT carbowax 20M fused silica capillary column (50m x 0.3 mm i.d.) deactivated with carbowax 20M. The analysis was accomplished using splitless injection at 250°C and a temperature program with an initial hold of 5 min at 90°C lbllowed by a 5 °/rain increase for 7 min. Helium was used as the carrier gas. Mass spectral identification of cyc!ohexanol was accomplishedwith a Hewlett Packard 5986A GC/MS system. The rate of formation of cyclohexanol by Coho Salmon microsomes was found to be linear for the first 60 min with a gradual decrease in the rate from 60 to 90 min. The reaction rate for cyclohexane hydroxylation in this enzyme system is about an order of magnitude less than that observed in reconstituted rabbit liver enzyme systems. 4's The optimum temperature range for the hydroxylation occurs between 15 and 25°C with a significant decrease outside this range (Fig. 1). The maximum rate determined at 20°C occurs at a substantially lower temperature than observed for mammalian systems and probably reflects genetic, developmental and environmental factors. 6 Cyclohexane hydroxylation reached a maximal rate between pH 8-0 and 8-5 (Fig. 2). This rate was nearly identical for incubations buffered with either TRIS or BICINE. Similar studies with rabbit liver microsomes 7 and yeast enzyme systems 8 demonstrated maximal activity at pH 7-4 to 7.6 and pH 6-8 to 7.0, respectively. The pH dependence in the salmon microsomal system would appear to require deprotonation of an amino acid side chain with a pKa of approximately 7. The maximal rate for the hydroxylation was found to be highly ionic strength dependent. In the range from 0-026 to 1-40, adjusted with the addition of KC1, the reaction rate dramatically
4.0
3.0
2.0
1.0
0
I
5
I
10
'i °
I
I
I
I
15
20
25
33
35
Fig. 1. Temperature dependence ofhydroxylation ofcyclohexane. The reaction mixture contained, per 2 ml of final volume: 0.5 ml of the microsomal preparation, 0.05 .~tTris-Cl buffer (pH 7.5), 0.1 M KCI, l-0mM NADPH, and 2 0 ~ glycerol. The incubations were carried out at the temperatures indicated. The reaction was initiated with the addition of 93 nmol of cyclohexane,
3.0
2.0
~_ 1.o
B 0
I
I
I
5
6
7
I
I
8
9
pH
Fig. 2. pH dependence of hydroxylation of cyclohexane. The reaction rate was determined at 20°C using MES (pH 5-5 to 6.0), HEPES (pH 7.5 to 8-0), TRIS (pH 7-5 to 8.0) and BICINE (pH 8.5 to 9.0) at 0.05 st.
132
John M. Kcnnish. Colleen Montova Jane Ve'hitsett, John S. French
increased below 0.50 with a maximum at 0.026. Studies in mammalian liver microsomal systems suggests optimal activity at 0-05 with a significant reduction at lower values of ionic strength. 8 During the course of these studies we observed gradual loss o f activity in the salmon microsomal system. More recent studies suggest a substantial reduction in this lost activity with storage at - 8 0 °C. We are investigating other methods of microsomal preparation in hopes of producing a more stable enzymatic system.
REFERENCES I. Coon, M. J., van der Hoeven, T. A., Dahl, S. B. & Haugen, D. A. Methods Enzymol., 52C, 109-17 (1978). 2. Bradford, M. Anal. Biochem., 72, 248-54 (1976). 3. Omura, T. & Sato, R. J. Biol. Chem., 239, 2370-8 (1964), 4. Coon, M. J., van der Hoeven, T. A., Haugen, D. A., Guengerich, F. P., Vermilion, J. L. & Ballou, D. P. In Cytochrome P-450andb-5(D. Y. Cooper, O. Rosenthal, R. Snyder & C. Witmer, eds), Plenum Publishing, New York, pp. 25-46, 1975. 5. Nordblum, G. D. & Coon, M. J. Arch. Biochem. Biophys., 180, 343-7 (1977). 6. Forlin, L., Andersson, T., Koivusaari, U. & Hansson, T. Marine Environ. Res., 14, 47-58 (1984). 7. Lu, A. Y., Junk, K. W. & Coon, M. J. J. Biol. Chem., 244, 3714-21 (1969). 8. Dttppel, W., Lebeault, J. & Coon, M. J. Eur. J. Biochem., 36, 583-92 (1973).
Metabolic Conversion of Cyclohexane by Pacific Salmon Microsomal Preparations John M. Kennish, Colleen Montoya, Jane Whitsett & John S. French Department of Chemistry, University of Alaska, Anchorage, Anchorage, Alaska 99508, USA
Hydrocarbons are being introduced into the marine environment from a variety of sources including combustion processes, crude oil spills, fuel oil spills, and controlled disposal such as processed ballast water. Even in the relatively clean waters of Alaska hydrocarbons are being released at an alarming rate. The purpose of this study was to determine the optimum conditions for the metabolism of a representative hydrocarbon by Coho Salmon Oncorhynchus kisutch liver microsomes. The product of cyclohexane metabolism in the salmon microsomal system was identified by gas chromatography-mass spectrometry as cyclohexanol. Conditions of the microsome incubation were varied systematically to determine the optimum temperature, pH, and ionic strength for cyclohexanol production. Cyclohexanol was quantified by capillary column gas chromatography. Maximum cyclohexanol formation was achieved at 20 ° C, a p H of 8.0-8.5 and an ionic strength of 0"026. A linear rate of cyclohexanol formation is seen from 0-60 rain of incubation and there is an apparent decrease in the rate from 60-90 rain. Poor stability of the microsomal preparation from the species studied was also identified and several stability studies have been undertaken using cyclohexane metabolism as a monitor. Adult Coho Salmon were caught by gillnet in Cook Inlet, Alaska. The fish were bled and the livers removed and stored on ice until processed. The livers were pooled and microsomes were prepared according to the method of Coon et al. 1 The microsomes were extracted with 0.25 M sucrose buffer to remove cytosolic factors and the final pellet was resuspended in 0-05 M Tris-C1 buffer (pH 7.5) containing 0.1 .~ KC1, 129 Marine Environ. Res. 0141-1136/85/$03-30 © Elsevier Applied Science Publishers Ltd,
England, 1985. Printed in Great Britain
130
John M. Kennish, Colleen Montoya, Jane Whitsett. John S. French
0-lmst EDTA, 0.01mM phenylmethane-sulfonyl fluoride. 0-01mM dithiothreitol and 20 ° o glycerol. The resuspended microsomes contained 19.1mgml of protein z and a P-450 concentration of 0.630/2M. 3 The microsomes were stored at - 3 0 ° C until used. Unless otherwise indicated, incubation mixtures were prepared in a 2 ml volume consisting of the microsomal preparation, 0.05.~I Tris-Cl buffer (pH 7.5), 0.1M KCI, 1.0mM NADPH, and ..~,'~n°//oglycerol. After addition of 10/21 ofcyclohexane the mixtures were incubated in a shaking water bath at constant temperature. The samples were extracted with 1.0ml of methylene chloride containing the internal standard (lhexanol). The organic phase was removed and concentrated to 50/21 with nitrogen. Analysis of cyclohexanol was accomplished using a Hewlett Packard 5840A gas chromatograph equipped with a flame ionization detector and a WCOT carbowax 20M fused silica capillary column (50m x 0.3 mm i.d.) deactivated with carbowax 20M. The analysis was accomplished using splitless injection at 250°C and a temperature program with an initial hold of 5 min at 90°C lbllowed by a 5 °/rain increase for 7 min. Helium was used as the carrier gas. Mass spectral identification of cyc!ohexanol was accomplishedwith a Hewlett Packard 5986A GC/MS system. The rate of formation of cyclohexanol by Coho Salmon microsomes was found to be linear for the first 60 min with a gradual decrease in the rate from 60 to 90 min. The reaction rate for cyclohexane hydroxylation in this enzyme system is about an order of magnitude less than that observed in reconstituted rabbit liver enzyme systems. 4's The optimum temperature range for the hydroxylation occurs between 15 and 25°C with a significant decrease outside this range (Fig. 1). The maximum rate determined at 20°C occurs at a substantially lower temperature than observed for mammalian systems and probably reflects genetic, developmental and environmental factors. 6 Cyclohexane hydroxylation reached a maximal rate between pH 8-0 and 8-5 (Fig. 2). This rate was nearly identical for incubations buffered with either TRIS or BICINE. Similar studies with rabbit liver microsomes 7 and yeast enzyme systems 8 demonstrated maximal activity at pH 7-4 to 7.6 and pH 6-8 to 7.0, respectively. The pH dependence in the salmon microsomal system would appear to require deprotonation of an amino acid side chain with a pKa of approximately 7. The maximal rate for the hydroxylation was found to be highly ionic strength dependent. In the range from 0-026 to 1-40, adjusted with the addition of KC1, the reaction rate dramatically
4.0
3.0
2.0
1.0
0
I
5
I
10
'i °
I
I
I
I
15
20
25
33
35
Fig. 1. Temperature dependence ofhydroxylation ofcyclohexane. The reaction mixture contained, per 2 ml of final volume: 0.5 ml of the microsomal preparation, 0.05 .~tTris-Cl buffer (pH 7.5), 0.1 M KCI, l-0mM NADPH, and 2 0 ~ glycerol. The incubations were carried out at the temperatures indicated. The reaction was initiated with the addition of 93 nmol of cyclohexane,
3.0
2.0
~_ 1.o
B 0
I
I
I
5
6
7
I
I
8
9
pH
Fig. 2. pH dependence of hydroxylation of cyclohexane. The reaction rate was determined at 20°C using MES (pH 5-5 to 6.0), HEPES (pH 7.5 to 8-0), TRIS (pH 7-5 to 8.0) and BICINE (pH 8.5 to 9.0) at 0.05 st.
132
John M. Kcnnish. Colleen Montova Jane Ve'hitsett, John S. French
increased below 0.50 with a maximum at 0.026. Studies in mammalian liver microsomal systems suggests optimal activity at 0-05 with a significant reduction at lower values of ionic strength. 8 During the course of these studies we observed gradual loss o f activity in the salmon microsomal system. More recent studies suggest a substantial reduction in this lost activity with storage at - 8 0 °C. We are investigating other methods of microsomal preparation in hopes of producing a more stable enzymatic system.
REFERENCES I. Coon, M. J., van der Hoeven, T. A., Dahl, S. B. & Haugen, D. A. Methods Enzymol., 52C, 109-17 (1978). 2. Bradford, M. Anal. Biochem., 72, 248-54 (1976). 3. Omura, T. & Sato, R. J. Biol. Chem., 239, 2370-8 (1964), 4. Coon, M. J., van der Hoeven, T. A., Haugen, D. A., Guengerich, F. P., Vermilion, J. L. & Ballou, D. P. In Cytochrome P-450andb-5(D. Y. Cooper, O. Rosenthal, R. Snyder & C. Witmer, eds), Plenum Publishing, New York, pp. 25-46, 1975. 5. Nordblum, G. D. & Coon, M. J. Arch. Biochem. Biophys., 180, 343-7 (1977). 6. Forlin, L., Andersson, T., Koivusaari, U. & Hansson, T. Marine Environ. Res., 14, 47-58 (1984). 7. Lu, A. Y., Junk, K. W. & Coon, M. J. J. Biol. Chem., 244, 3714-21 (1969). 8. Dttppel, W., Lebeault, J. & Coon, M. J. Eur. J. Biochem., 36, 583-92 (1973).