I'm working on the impact of water pollution on the physiological and histopathological responses of Nile tilapia.
In the attachment you can find a protocol that might help you with your project.
Cheers, Christian
Article Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase
Hi Afaf,
As a state-of-the-art paper of my fellow Fred Guengerich and his colleagues concerns exclusively determination of P450s in the rodent and human samples, let me suggest you a protocol related directly to the procedure with P450-containing samples from a fish, – the procedure established in the lab (now Institute) of John Stegeman.
Fish livers were thawed and homogenized (approximately 1g liver tissue) in for volumes of homogenization buffer (0,08M Na2HPO4; 0.02M KH2PO4; 0.15M KCl, pH 7.4). Homogenate was centrifuged at 10.000 x g for 20 min at 4°C. The supernatant was ultracentrifuged at 100.000 x g for 60 min at 4°C. The pellet was resuspended in 1ml of resuspension buffer (homogenization buffer with 20% glycerol, v/v) to obtain the hepatic microsomal fraction. All the spectroscopic P450 (thereafter – CYP) analyses were carried out with this fraction. Protein concentration was determined using bovine serum albumin (BSA).
Analysis of total CYP was based on the methods described by Omura and Sato, using differential visible spectroscopy with some adaptations. This protocol compares an oxidized aliquot of microsomal fraction with another reduced aliquot, which is bubbled with carbon monoxide. Liver perfusion was performed with an isotonic saline solution (0.9%) in order to prevent contamination by hemoglobin. Because fish liver samples of are usually very small, the perfusion was carried out by injecting the isotonic solution into each fragment of tissue until it showed a yellowish color.
Blood of a fish of interest was collected by cardiac puncture in order to obtain hemoglobin. The hemoglobin was treated exactly as the microsome samples and UV-vis spectra were recorded. Aiming at reducing the influence of hemoglobin on CYP spectra, concentrations of 20 μM of phenasine methosulphate (PMS) and 2 mM sodium ascorbate were tested, which have been suggested to reduce contaminant hemoglobin without reducing CYP. Analysis was carried out in a dual beam differential spectrophotometer.
Best wishes,
Ilya
I am using the spectral method as shown exactly in the attached reference by Christian and it worked very well.
Regards.
Zoheir
can we use single beam spectrophotometer instead of dual beam differential spectrophotometer? If yes please let me know the procedure details or attach a research article if any available?
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