Microplate Assay Measurement of Small Amount of P450

Abstract

Cytochrome P450 (P450) has the feature of having an absorption spectrum in the 450 nm if it binds with carbon monoxide (CO). A spectrophotometrical assay of the P450 content was established through the use of this feature. We improved the analytical methodology to deal with a small sample and to become suitable for a high-throughput measurement. A microplate-based assay using a CO releasing agent, dimanganese decacarbonyl (DMDC), and an exposure to light, instead of a CO bubbling was the process utilized. The absorbance difference between the sample which added DMDC and did not add DMDC was measured after adding a sodium hydrosulfite solution to each sample. The absorbance index was determined on the basis of the optical path length of the microplate well. The P450 content was calculated using the absorbance difference and this absorbance index. A suitable concentration of DMDC solution, lighting time and luminescence intensity were determined. The P450 density of rats liver microsomes determined by this new method had a good correlativity to the traditional method. Analysis of P450 content in the microsome of the liver and intestine are necessary as a predictor of a drug-interaction which are referred from induction or down-regulation of P450 at an early phase of development of functional foods or drugs. The new assay we propose was suitable for a small sample and high-throughput analysis of multiple samples, and suitable to determine influences exerted by a test sample which was administered to small animals such a rodent.

Keywords:

• Cytochrome
• P450
• Dimanganese decacarbonyl
• Microplate
• Microsome

Notes

Note. Protein concentration of liver microsome and DMDC concentration was 2.0 mg/mL and 20 mM, respectively. Lighting time was 4 minutes. The luminous intensity of >20000 lux was beyond the range of the illuminance meter when the illuminating board was set directly on a microplate. Setting a microplate directly on the illuminating board was suitable for the method of light irradiation.

Note. Each microsome of liver and intestine was prepared with 2.0 mg/mL of protein concentration. After adding 2 µL DMDC at 20 mM, the illuminating board was set directly on a microplate for 0–10 minutes. Results are expressed as means ±SD (n = 5). The microsomal P450 density reached a maximum after light irradiation for 4 minutes.