APPENDIX

Total Cytochrome P450

Garfinkel (1958) and Klingenberg (1958) independently showed that liver microsomes contained a haemoprotein which upon reduction was capable of combining with carbon monoxide to produce an intense absorption band at 450nm. This carbon monoxide-liver pigment complex gave rise to the term 'P450' (Omura and Sato, 1962), a terminology which was subsequently changed to 'cytochrome P-450' when the pigment was shown to be a haemoprotein.

The haemoprotein, cytochrome P-450, appears to exist in high and low spin states. The spin states correlate well with optical spectra. In general, haemoproteins with Soret bands near 390nm are high spin haemoproteins, whereas, haemoproteins with Soret bands around 420nm are low spin haemoproteins. The use of optical spectra studies applied to cytochrome P450 has permitted considerable extrapolation of various studies on the interaction of oxygen, reducing agents, inhibitors, substrates and other molecules with cytochrome P450 in an attempt to produce a theoretical explanation of the nature of the haemoprotein-molecular interactions.

In some instances, the addition of substrate or certain other molecules to the mixture of high spin and low spin cytochrome P450 results in a transition of some of the low spin cytochrome P450 into high spin haemoprotein. A large number of reagents and enzymes react with cytochrome P450 to produce a related but biologically inert haemoprotein termed 'cytochrome P420'. This haemoprotein is given this name because the reduced-carbon monoxide difference spectrum of cytochrome P420 has an intense absorption band at 420nm. The reagents which effect the change from cytochrome P450 to cytochrome P420 include phospholipases, certain alcohols, ketones, sulphydryl reagents, detergents and many other substances.

Molecules which interact with the oxidized form of cytochrome P450 to produce enzyme substrate complexes with an absorption maximum around 390nm are termed Type-1 compounds and those that interact with the oxidized form of the haemoprotein to produce an absorption maximum around 420nm are termed Type-2 compounds. Therefore, Type-1 co-ordination complexes are in fact high spin complexes while the Type-2 co-ordination complexes are low spin complexes.

The hexa-coordinated low-spin or penta-coordinated high spin states are descriptions of the electronic shells around the iron atom. When cytochrome P450 binds a substrate, there is a pertubation of these electronic shells and the haem iron changes from its hexa-coordinated to the penta-coordinated state. For a reaction to occur between cytochrome P450 and oxygen or carbon monoxide the haem iron must be reduced from the ferric to its ferrous state so that these molecules may bind to the haem iron. Reduction of the ferric to ferrous form of the haemoprotein is achieved by the addition of sodium dithionite to sample and reference cuvettes, and carbon monoxide gassed through sample cuvettes only. The reduced-CO versus reduced difference spectrum is used to measure total cytochrome P450 in a sample. Method is that of Omura and Sato (1964).

Reagents

0.1M Potassium phosphate buffer pH 7.6
Sodium dithionite
Carbon monoxide gas (store and use in fume hood)

Method

0.5ml of 25% microsomes are added to 2.5ml of 0.1M Potassium phosphate buffer. A few crystals of sodium dithionite are added, mixed and divided into two cuvettes. Auto zero and run baseline between 400 to 500nm using a split-beam spectrophotometer. The sample cuvette is saturated with about 30 to 40 bubbles of CO at a rate of about 1 bubble per second. Run sample (note curve number). Go to Methods and Peak detect. Enter curve number and display. Make a hard copy and return to Methods for a new sample.

Calculation

The absorbance at 490nm (isobestic point) serves as a reference point.
The abs. diff. = the difference in optical density between 450 and 490nm
The extinction coefficient = 91mM-1cm-1 = 91µmol/ml for 1cm path length
Cytochrome P450 content (nmol/g liver)
= [(abs.diff. x 1000)/91] x dilution factor x 25% microsomes
=[(abs.diff. x 1000)/91] x 6 x 4
dilution factor: 0.5ml of 25% microsomes in a total volume of 3ml
= 1 in 6 dilution.
[(nmol/g liver)/(mgPr/g liver)] = nmol/mgPr.


p-nitrophenol hydroxylase

p-nitrophenol is metabolised in the uninduced liver by glucuronide and sulphate conjugated pathways. However, it is rapidly metabolised to 4-nitrocatechol in ethanol-treated rats. Ethanol is known to induce P450 IIE1.

Under alkaline conditions the product 4-nitrocatechol can be measured at a wavelength of 536nm because whilst both p-nitrophenol and 4-nitrocatechol have absorbance maxima at around 350-400nm, only 4-nitrophenol has an absorbance maximum at around 550nm (Reinke and Mayer, 1985). NADPH was found to be the preferred co-factor for p-nitrophenol hydroxylation by hepatic microsomes from ethanol-treated rats. NADH supported rates that were 13% of the activity observed with the NADPH-generating system and the reaction was subject to substrate inhibition at high concentrations of p-nitrophenol ( Chrastil and Wilson, 1975).

Reagents

0.2M Potassium phosphate buffer, pH 6.8
1mM Ascorbate (17.6mg/100ml)
1mM p-nitrophenol (13.9mg/100ml)
0.5mM nitrocatechol (7.8mg/100ml)
10mM NADPH (8mg/ml in 1% NaHCO3)
10M NaOH
0.6N ice-cold perchloric acid

Method

The hydroxylation of p-nitrophenol was followed essentially by the method of Reinke and Mayer, (1985) as modified by McCoy and Koop, (1988).

Table

Both the standard and samples are incubated for 3min. at 37ºC in a shaking water bath, but NADPH is only added to the samples to initiate the transformation of p-nitrophenol to 4-nitrocatechol, but not the standard. After a 10min. incubation at 37ºC, the reaction is terminated by the addition of 0.5ml ice-cold perchloric acid. This lowers the pH, the colour disappears and the proteins in the microsomes denature. The protein is precipitated by centrifugation at 3000rpm for 10min. 1ml of the supernatant is then added to 0.1ml of 10M NaOH in a cuvette. The absorbance is read immediately at 536nm since the colour fades with time. Concentration of 4-nitrocatechol formed is read from the standard curve.
Ascorbate in the reaction mixture prevents p-nitrophenol from forming a quinone. NaOH ionizes the phenolic hydroxyl groups (pKa = 10.1).

Calculation

50µl (0.05ml) of 25% microsomal supernatant gives a factor of = 4 x 20
Incubation time of 10min gives a factor of = 6
Therefore, Total factor = 4 x 20 x 6 = 480
Amount of sample (nmol) x 480 = nmol/g liver/hr
[nmol/g liver/hr]/[mgPr/g liver] = nmol/mgPr/hr


Ethoxyresorufin O-deethylase

Some substrates appear to be relatively specific for PAH - inducible cytochrome P450. Burke and Mayer (1975) demostrated that an alkylphenoxazone derivative, 7-ethoxyresorufin, was highly specific for the haemoprotein P450 IA1. Subsequent work has demonstrated in humans, mice, rabbits and rats that PAH-induced cytochrome P450s specifically utilize this phenoxazone ether as a substrate (Norman et al., 1978; Lang and Nebert, 1981; Guengerich et al., 1982; McManus et al., 1990). The assay method used is the direct fluorimetric assay by Burke and Mayer (1974). Fluorometry is based on the principle that many compounds absorb light and then immediately re-emit some of the energy as light of a longer wavelength. The emitted fluorescence light is observed at 90µ to the incident light. Two wavelength selectors are required, one to transmit the desired excitation wavelength and the other to select the desired emmission wavelength. Fluorometry can be extremely selective since only certain wavelengths of light will excite a given compound. Similarly, fluorescence will occur only at certain wavelengths. In other words, fluorescent compounds have a characteristic excitation spectrum and a characteristic fluorescent spectrum. Ethoxyresorufin and resorufin are intensely fluorescent. The excitation and emission maxima for both compounds lie in the visible spectrum. The assay method measures the fluorescence of resorufin which is the product of the reaction.

Table The excitation wavelength is set at 510nm instead of 560nm because microsomal turbidity increases the excitation light-scatter.

Reagents

0.1M Tris buffer pH 7.8 (in water bath at 37ºC)
0.53mM Ethoxyresorufin (253µg/2ml DMSO)
50µM NADPH (41.7mg/ml in 1%NaHCO3)
0.1mM Resorufin
1%NaHCO3

Instrument

Perkin-Elmer L5 Luminescence Spectrometer
Perkin-Elmer 561 Recorder

Method

The general reaction mixture, prepared in a fluorimeter cuvette, contained:

0.1M Tris buffer pH 7.8			2ml
25% microsomes				50µl
0.53mM Ethoxyresorufin			3µl
A baseline of fluorescence was recorded at an excitation wavelength of 510nm and an emission wavelength of 586nm, with slit arrangement of 10 (excitation) and 2.5 (emission). A 10µl aliquot of NADPH (50µM, Sigma Chemicals) was stirred into the mixture to start the reaction and the progressive increase in fluorescence, as ethoxyresorufin was de-ethylated to resorufin, was recorded with a Perkin-Elmer chart recorder.

Calibration

The fluorimeter was calibrated with:

0.1mM resorufin				10µl
25%microsomes				50µl
0.1M Tris buffer pH 7.8			2.0ml
Since the fluorescence emitted by one substance may be absorbed or quenched by other substances in the reaction mixture, it was appropriate to add microsomes and buffer to the calibration sample so as to eliminate the need for an internal standard and a blank.

Calculation


Pentoxyresorufin O-depentylase


Erythromycin N-demethylase

Erythromycin A (a macrolide antibiotic).

Erythromycin is rapidly demethylated by microsomal enzymes to yield des-N-methyl-erythromycin and formaldehyde.

Many drugs containing N-methyl or O-methyl groups undergo demethylation by the microsomal enzymes to give formaldehyde and demethylated compounds ( Brodie et al., 1958). Since Erythromycin A contains both N-methyl and O-methyl groups, Mao and Tardrew (1965) did an experiment to ascertain whether formaldehyde is released by demethylating N-methyl or O-methyl groups or both. They did found out that formaldehyde comes exclusively from the dimethyl amino group of desosamine.

Reagents

  1. Formaldehyde standard (HCHO, M.W. = 30)
    Standard required = 3µmol/ml = 3mM
    1M = 30g/l; 3mM = 30 x 3 x10-3 g/l = 90mg/l = 9mg/100ml
    Formaldehyde solution is 40%
    40g in 100ml; 1g in 2.5ml; 90mg in 225µl; 9mg in 22.5µl
    Take 22.5µl of 40% HCHO and make up to 100ml giving 3mM (3µmol/ml).
    Standard stored at 3µmol/ml
    Dilute standard 1 in 3 giving 1µmol/ml.
  2. Nash reagent:
    	Ammonium acetate			30g
    	Acetylacetone (2,4-pentanedione)	0.4ml
    	Glacial acetic acid			0.6ml
    	make up to 100ml.
    
  3. 50mM Potassium phosphate buffer, pH 7.25
  4. Erythromycin (M.W. = 734); 10mM = 147mg in 20ml.
  5. MgCl2 (M.W. = 203); 150mM = 3g in 100ml.
  6. NADPH (M.W. =833); 10mM = 8mg in 1ml of 1% NaHCO3
  7. 12.5% TCA (trichloroacetic acid) w/v
Method

The microsomal erythromycin N-demethylase activity was performed as described by Wrighton et al., (1985). To duplicate test tubes the following were added:

	50mM Potassium phosphate buffer, pH 7.25		0.6ml
	Magnesium chloride (150mM)				0.1ml
	Erythromycin (10mM)					0.1ml
	Microsomal suspension (25%)				0.1ml
The tubes were pre-incubated for 3min. at 37ºC and the reaction initiated by the addition of 0.1ml NADPH (10mM). After a further 10min. incubation the reaction was terminated by the addition of 0.5ml of ice-cold 12.5%(w/v) trichloroacetic acid. The tubes were centrifuged at 3000rpm for 10min to remove the protein. 1ml of the supernatant was then added to 1ml of freshly prepared NASH reagent. The tubes were heated in a water bath at 50ºC for 30min and after cooling the absorbance was read at 412nm. The erythromycin N-demethylase activity was calculated from standards (0-100µM formaldehyde) which were run in parallel.

Calculation

0.1ml of 25% microsomal supernatant gives a factor of = 4 x 10 = 40
Incubation time = 10min.; Therefore, time factor = 6; Total factor = 40 x 6 = 240
amount of sample (nmol x 240) = nmol/g liver/hr
(nmol/g liver/hr)/(mgPr/g liver) = nmol/mgPr/hr


Lauric acid hydroxylase

P450IVA1 when induced causes 12-hydroxylation of lauric acid, otherwise lauric acid is 11-hydroxylated. 14C-lauric acid is hydroxylated at the w and w-1 positions. These are solvent extracted and separated by TLC. Measurement of the radioactive fraction yields the required results. ( Parker and Orton, 1980).

Reagents

500mM Tris-HCl buffer, pH 7.4
1mM Lauric acid
10µCi ml-1 14C-lauric acid
40mM NADPH (33mgml-1 in 1% NaHCO3)
3M HCl
Ether

Method

Incubation system is prepared in Sovirel tubes (polyethylene tubes).
In each tube add:

Tris-HCl buffer (500mM), pH 7.4			1.5ml
Lauric acid (1mM)				0.2ml
14C-lauric acid (10µCi/ml)			0.1ml
25% microsomes					0.2ml
Incubate at 37ºC for 5min. Add 40µl NADPH to sample tubes but not to blanks.
Lauric acid: make 40mg/ml in methanol = 200mM
Take 50µl in 9.95ml Tris = 1mM.
40mM NADPH: 33mg/ml in 1% NaHCO3. After addition of NADPH incubate for 10min. Terminate by addition of 3M HCl -200µl. Add 10ml ether from dispenser. Shake on rotary shaker for 10min - use caps. Stand for 5min. Pipette 7.8ml of upper layer. Evaporate to dryness under nitrogen. Add 60µl methanol to tubes (mix). Spot 25µl unto silica gel plates (without fluorescence). Plates developed in a hexane:diethylether:acetic acid system (70:28:1). The plates are then scanned with a Berthold TLC plate scanner.

Calculation

0.2ml of 1mM lauric acid used. In 1000ml there is 1mmol.
In 0.2ml there will be (0.2ml x 1 x 10-3mol)/1000ml=0.2 x 10-6mol=0.2µmol=200nmol.
In 0.2ml of lauric acid there is 200nmoles. 200nmoles is equivalent to 100% LA
Therefore, %LAOH = (%LAOH x 200nmoles)/100%LA = nmoles
200µl of 25% microsomes used = 5 x 4 dilution factor.
Incubation time = 10min.
nmoles/g liver/min = (nmoles x 5 x 4)/10min
nmoles/mgPr/min = (nmoles/g liver/min)/(mgPr/g liver)


Cyanide-insensitive palmitoyl-CoA oxidation

Reagents

A: 60mM Tris-HCl, pH 8.3
B: 1%(v/v) Triton X-100 in 60mM Tris buffer
C:	 75µM CoA			(5.8mg)
	180µM FAD			(14.9mg)
	555µM NAD			(37.0mg)
	141mM Nicotinamide		(1720mg)
	4.2mM DTT			(64.8mg)
	3.0mM KCN			(19.8mg)
	255µg/ml BSA(fatty acid free)	(25.5mg)
All in 100ml Tris-HCl buffer, pH 8.3
D: 7.5mM Palmitoyl-CoA (20µl into 3ml)
Method and Calculation

Dilution factor = 10µl of 25% microsomes in cuvette = 100 x 4 = 400
Extinction coefficient = 6.22mM-1cm-1 at a wavelength of 340nm
6.22 = 1mM in 1000ml = 1µmol/ml = 1000nmol/ml
nmol/min = [Activity (Abs/min) x 1000 (nmol/ml)/6.22] x 400

Method and Calculation for Protein

In one set of test tubes put:
	25% microsomes (homogenate)		0.1ml
	0.5M NaOH				9.9ml
						10ml
Take 0.25ml from above (first set of tubes) into two set of test tubes and add
0.5ml of 0.5M NaOH to make 0.75ml
Dilution factor = 4 (25% microsomes) x 100 (0.1ml in 10ml) x 3 (0.25ml in 0.75ml)
= 4 x 100 x 3 = 1200
1/1200 = 0.83 x 10-3
µgPr in 0.83 x 10-3 g liver
mgPr in 0.83 g liver
In 1g liver there will be XmgPr/0.83g liver = XmgPr x 1.2 per g liver
µgPr x 1.2 = mgPr/g liver


Protein determination

An estimation of microsomal protein content is required as the assays are normally expressed as per unit protein. The spectrophotometric method uses intrinsic (ultra violet) chromophores such as aromatic residues (280nm) or peptide bonds (215nm) or by treatment with a reagent to yield a coloured or fluorescent product.

Lowry method

Initially involves:

  1. complexing the protein with Cu2+ in an alkaline solution. The complex is between Cu2+ and four nitrogen atoms, two from each of two adjacent peptide chains.
  2. In addition, the copper appears to catalyse the reduction, by the tyrosine and tryptophane residues, of the phosphomolybdate/phosphotungstate anions in the Folin phenol reagent, added subsequently. This latter reaction leads to a blue colour which can be measured at 700 - 750nm. Cu++ ions appear to be the catalyst in this reaction.
Reactions:
  1. Formation of the protein - copper complex.
  2. Reduction of the phosphomolybdate - phosphotungstate reagent (Folin - Ciocalteu phenol reagent) by tyrosine and tryptophan residues.
Reagents: