Abstract
Cytochrome P-450 (P-450) isozymes are well separated by high-performance liquid chromatography with an ion-exchange column using buffer containing 0.4% Emulgen 911 (polyphenyl ether) as a detergent. However, it is impossible to monitor the elution profile of protein at 280 nm in the presence of Emulgen. Thus, we have developed a technique for determining the elution profile of protein in the presence of Emulgen. The absorption maximum and minimum of Emulgen 911 were at 276 and 244 nm, respectively. At 244 nm, the elution profile of 0.5 nmol (35 μg) of P-450 was detected as a peak in the presence of 0.4% Emulgen 911. The peak area increased linearly with increasing amount of P-450 up to 5 nmol (350 μg). Identification and estimation of the purity of the eluted P-450 are possible by dual monitoring at 244 and 417 nm, the wavelength for the detection of hemoprotein.
