Abstract
This chapter discusses the isolation and purification of bacterial cytochrome P-450. Cytochrome P-450cam content in the whole cells is monitored by difference optical-absorbance spectrophotometry using an Aminco Model DW-2 recording spectrophotometer in the split-beam mode. A 6 ml aliquot of the cell suspension is gently deoxygenated by bubbling with argon, solid sodium dithionite is added to totally reduce the enzyme, the cell suspension is evenly divided into sample and reference cuvettes (1 cm pathlength), and a base line is recorded from 400 to 500 nm. The amounts of putidaredoxin reductase, putidaredoxin, and NADH are chosen so that cytochrome P-450cam is the rate-limiting component in the system. An identical specific activity is obtained when oxygen consumption is monitored in this system following the procedure previously outlined. The isolated cytochrome P-450cam is a homogeneous protein composed of a single polypeptide chain of molecular weight 43,900–45,000 as determined from the following: (1) molecular sieve chromatography, (2) equilibrium ultracentrifugation, and (3) 0.1% sodium dodecyl sulfate-10% polyacrytamide slab gel electrophoresis.
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