Highly Purified Liver Microsomal Cytochrome P450: Properties and Catalytic Mechanism

Recent studies in this laboratory on two forms of cytochrome P450 purified to homogeneity from rabbit liver microsomes are reviewed. The two forms, phenobarbital-inducible P450LM2 and 5,6-benzoflavone-inducible P450LM4, differ in subunit molecular weight, identity of the C-terminal amino acid, optical and EPR spectra, and other properties. As isolated, oxidized P450LM2 is in the low spin state, whereas P450LM4 is largely, but non entirely, in the high spin state. Mechanistic studies have shown the following: (a) P450LM2 may accept two electrons, calculated per heme, from dithionite or NADPH in the presence of catalytic amounts of the reductase, and may donate two electrons to various oxidizing agents, including molecular oxygen. (b) Hydrogen peroxide is formed in the reconstituted system in the presence of NADPH and oxygen, and the amount varies with the substrate added. (c) Hydrogen peroxide and other hydroperoxides apparently donate the oxygen atom inserted into substrate during hydroxylation in the absence of 0 2 and an external donor. (d) Stopped flow spectrophotometry has provided evidence for two distinct oxygenated complexes of the reduced cytochrome. The reductase and cytochrome b5 may play an effector role in increasing the rate of decomposition of the second complex during oxygen insertion into substrate. A scheme is proposed for the mechanism of action of purified P450LM2, based on these and other findings

Highly Purified Liver Microsomal Cytochrome P450: Properties and Catalytic Mechanism

Recent studies in this laboratory on two forms of cytochrome P450 purified to homogeneity from rabbit liver microsomes are reviewed. The two forms, phenobarbital-inducible P450LM2 and 5,6-benzoflavone-inducible P450LM4, differ in subunit molecular weight, identity of the C-terminal amino acid, optical and EPR spectra, and other properties. As isolated, oxidized P450LM2 is in the low spin state, whereas P450LM4 is largely, but non entirely, in the high spin state. Mechanistic studies have shown the following: (a) P450LM2 may accept two electrons, calculated per heme, from dithionite or NADPH in the presence of catalytic amounts of the reductase, and may donate two electrons to various oxidizing agents, including molecular oxygen. (b) Hydrogen peroxide is formed in the reconstituted system in the presence of NADPH and oxygen, and the amount varies with the substrate added. (c) Hydrogen peroxide and other hydroperoxides apparently donate the oxygen atom inserted into substrate during hydroxylation in the absence of 0 2 and an external donor. (d) Stopped flow spectrophotometry has provided evidence for two distinct oxygenated complexes of the reduced cytochrome. The reductase and cytochrome b5 may play an effector role in increasing the rate of decomposition of the second complex during oxygen insertion into substrate. A scheme is proposed for the mechanism of action of purified P450LM2, based on these and other findings

A specific radioimmunoassay for androstenedione with reduced bridge-binding

Antibody used in a steroid radioimmunoassay raised against a steroid hapten-carrier protein conjugate may recognize both the hapten and the chemical bridge to the protein. Use of the same bridge in the radioisotopic label may lead to higher affinity binding to the label than to the native steroid. Inhibition curves under these conditions are shallow and generally not acceptable for radioimmunoassay procedures. We have developed a radioimmunoassay for androstenedione that employs different bridges at the 11[beta] position of the steroid for the protein conjugate and label. The resulting assay has greatly reduced bridge-binding, has an acceptable slope for the standard curve and is very specific as evidenced by low crossreactivies to other steroids.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24709/1/0000130.pd

A chemical approach to solving bridging phenomena in steroid radioimmunoassays

Steroid radioimmunoassays (RIA) employ antibodies raised against a carrier protein-steroid conjugate. Individual antibodies may recognize the steroid, the protein or the chemical bridge used to Join them together. Use of the same bridge In the tracer results in higher affinity binding of the tracer than the native ligand which in turn results in a loss of sensitivity and precision. We have greatly reduced bridge-binding In a RIA for androstenedione. Conjugates and radioiodinated labels were prepared with either an ester op ether chemical bridge. By using an antibody and the corresponding label with the heterologous bridge very sensitive assays were obtained.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24289/1/0000555.pd

The measurement of testosterone and oestradiol-17[beta] using iodinated tracers and incorporating an affinity chromatography extraction procedure

The development of sensitive radioimmunoassays (RIA) for testosterone and oestradiol-17[beta], utilising 125I-radioligands, is described. Use of an homologous bridge at the same site of attachment, for both the radioligand and the steroid-carrier protein conjugate employed in raising antibodies, normally results in a loss of assay sensitivity and precision. This was overcome in the oestradiol assay by utilising an heterologous configuration at the site of attachment (11[alpha] vs 11[beta]). In contrast, for testosterone, even though an homologous bridge and site of attachment was used for the radioligand and the steroid-carrier protein conjugate, a very sensitive assay with extremely high antibody titres (dilution of 1:2 x 106) was achieved. This finding was repeated with a different antiserum suggesting that the "bridge binding" phenomenon may be related to the position of attachment to the steroid molecule. In addition, an antibody-Sepharose 4B affinity chromatography extraction procedure has been developed for both oestradiol and testosterone. This approach allows the measurement of very low concentrations of steroids from large volumes of a variety of biological fluids. As antibody-linked Sepharose 4B uses high concentrations of antibody, steroids of similar structure are extracted from biological fluids. However, the cross-reactivity of these related steroids are very low in the RIA's, ensuring good specificity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25490/1/0000031.pd