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Measurement of Total Cytochrome P450 Content in Digestive Gland Microsomes of Mussel (Mytilus sp.)
449
Technical Annex (Chapter 1) Measurement of Total Cytochrome Microsomes of Mussel (Mytilus sp.)
P450
Content
in Digestive
Gland
D.R. Livingstone, L.D. Peters
Plymouth Marine Laboratory, Citadel Hill, Plymouth PL 1 2PB, United Kingdom
Background:
Increases in total cytochrome P450 content of digestive gland microsomes of M. edulis has been observed both with experimental (Livingstone et al., Aquat. Toxicol. 7, 79-91, 1985 ; Livingstone, Mar. Ecol. Prog. Ser. 46, 37-43, 1988) and field (So16 et al. Comp. Biochem. Physiol. 113C, 257-265, 1996) exposure to polycyclic aromatic hydrocarbons and related compounds. Principle 9
Total cytochrome P450 content is measured by the carbon monoxide-difference spectrum of sodium dithionite reduced sample by a modification of the original method of Omura and Sato. Full details of the nature of the spectra obtained, and the uncertainties in the use of the alternative dithionite-difference spectra of carbon monoxide-treated sample are given in Livingstone et al. (Xenobiotica, 19, 10411062, 1989). Materials 9
1. Microsomal sample (approx. 10 mg protein ml-1 in 10 mM Tris-HC1 pH 7.6 containing 20 % w/v glycerol) -prepare as described in Livingstone (Mar. Ecol. Prog. Ser. 46, 37-43, 1988) and use either immediately, or after short-term storage (1-7 days) in liquid nitrogen. Store on ice while in use. 2. 100mM Tris-HC1 pH 7.6 (25 ~ 3. Distilled and deionized water 4. Fresh sodium dithionite 5. Carbon monoxide cylinder fitted with needle valve with rubber tube and glass pasteur pipette attached. Keep end of pipette in distilled water and set bubble rate to about 70 bubbles min-1. 6. Double-beam spectrophotometer, or single beam spectrophotometer with capacity for stor~g and manipulating spectra. 7. 1 cm path-leng~t~ semi-micro glass or plastic cuvettes.
450
M e t h o d for double-beam spectrophotometer :
1. Mix 1 ml of 100 mM Tris-HC1 Ph 7.6, 150 ml microsomes and 0.85 ml water and divide approximately equally between two cuvettes. Place one cuvette in reference beam of spectrophotometer. 2. Bubble carbon monoxide (approx. 70 bubbles over 1 minute) into second cuvette and place in sample beam of spectrophotometer. Scan from 500 to 400 nm at approximately 2nm min-1. Apply base-line correction and rescan to check for flat baseline. 3. Add a few grains of sodium dithionite to each cuvette, quickly mix and scan spectra 3 to 4 times over about 5 minuttes until spectral peaks have reached their maximum heights. Method for single-beam spectrophotometer 9
1.mix 0.5 ml of 100 mM Tris-HC1 pH 7.6, 75 ml microsomes and 0.425 ml water in a single cuvette. 2. Bubble carbon monoxide (approx. 70 bubbles over 1 minute) into cuvette and place in spectrophotometer. Scan from 500 to 400 nm at approximately 2 nm min1. Store spectrum. 3. Add a few grains of sodium dithionite to cuvette, quickly mix and scan spectra 3 to 4 times over about 5 minutes until spectral peaks have reached their maximum heights. Substract stored spectrum from stage 2 to obtain difference spectrum. Calculation :
1. Determine position of cytochrome P450 lmax (usually varies from about 448452 nm) and position of baseline which may be horizontal (situation A) or sloping (situation B). In the latter case, draw in the linear sloping baseline. 2. Calculate lmax peak absorbance (Abs) from either (lmax - 409 nm) (situation A) or (lmax - sloping baseline at lmax). 3. Calculate total cyt0chome content in pmol mg-1 microsomal protein from : Abs (OD units) x 13.3 91 x protein conc (mg ml-1) where 91 is ext. coeff, of cytochrome P450 in mM cm-1.
Technical Annex (Chapter 1) Measurement of Total Cytochrome Microsomes of Mussel (Mytilus sp.)
P450
Content
in Digestive
Gland
D.R. Livingstone, L.D. Peters
Plymouth Marine Laboratory, Citadel Hill, Plymouth PL 1 2PB, United Kingdom
Background:
Increases in total cytochrome P450 content of digestive gland microsomes of M. edulis has been observed both with experimental (Livingstone et al., Aquat. Toxicol. 7, 79-91, 1985 ; Livingstone, Mar. Ecol. Prog. Ser. 46, 37-43, 1988) and field (So16 et al. Comp. Biochem. Physiol. 113C, 257-265, 1996) exposure to polycyclic aromatic hydrocarbons and related compounds. Principle 9
Total cytochrome P450 content is measured by the carbon monoxide-difference spectrum of sodium dithionite reduced sample by a modification of the original method of Omura and Sato. Full details of the nature of the spectra obtained, and the uncertainties in the use of the alternative dithionite-difference spectra of carbon monoxide-treated sample are given in Livingstone et al. (Xenobiotica, 19, 10411062, 1989). Materials 9
1. Microsomal sample (approx. 10 mg protein ml-1 in 10 mM Tris-HC1 pH 7.6 containing 20 % w/v glycerol) -prepare as described in Livingstone (Mar. Ecol. Prog. Ser. 46, 37-43, 1988) and use either immediately, or after short-term storage (1-7 days) in liquid nitrogen. Store on ice while in use. 2. 100mM Tris-HC1 pH 7.6 (25 ~ 3. Distilled and deionized water 4. Fresh sodium dithionite 5. Carbon monoxide cylinder fitted with needle valve with rubber tube and glass pasteur pipette attached. Keep end of pipette in distilled water and set bubble rate to about 70 bubbles min-1. 6. Double-beam spectrophotometer, or single beam spectrophotometer with capacity for stor~g and manipulating spectra. 7. 1 cm path-leng~t~ semi-micro glass or plastic cuvettes.
450
M e t h o d for double-beam spectrophotometer :
1. Mix 1 ml of 100 mM Tris-HC1 Ph 7.6, 150 ml microsomes and 0.85 ml water and divide approximately equally between two cuvettes. Place one cuvette in reference beam of spectrophotometer. 2. Bubble carbon monoxide (approx. 70 bubbles over 1 minute) into second cuvette and place in sample beam of spectrophotometer. Scan from 500 to 400 nm at approximately 2nm min-1. Apply base-line correction and rescan to check for flat baseline. 3. Add a few grains of sodium dithionite to each cuvette, quickly mix and scan spectra 3 to 4 times over about 5 minuttes until spectral peaks have reached their maximum heights. Method for single-beam spectrophotometer 9
1.mix 0.5 ml of 100 mM Tris-HC1 pH 7.6, 75 ml microsomes and 0.425 ml water in a single cuvette. 2. Bubble carbon monoxide (approx. 70 bubbles over 1 minute) into cuvette and place in spectrophotometer. Scan from 500 to 400 nm at approximately 2 nm min1. Store spectrum. 3. Add a few grains of sodium dithionite to cuvette, quickly mix and scan spectra 3 to 4 times over about 5 minutes until spectral peaks have reached their maximum heights. Substract stored spectrum from stage 2 to obtain difference spectrum. Calculation :
1. Determine position of cytochrome P450 lmax (usually varies from about 448452 nm) and position of baseline which may be horizontal (situation A) or sloping (situation B). In the latter case, draw in the linear sloping baseline. 2. Calculate lmax peak absorbance (Abs) from either (lmax - 409 nm) (situation A) or (lmax - sloping baseline at lmax). 3. Calculate total cyt0chome content in pmol mg-1 microsomal protein from : Abs (OD units) x 13.3 91 x protein conc (mg ml-1) where 91 is ext. coeff, of cytochrome P450 in mM cm-1.