Cytochromes P-450 PB3a and PB3b, which appear to be equivalent to forms b and e described by Ryan et al. [Ryan, D.E., Thomas, P.E., & Levin, W. (1982) Arch. Biochem. Biophys. 216, 272-288], have been shown to share 97% sequence homology [Suwa, Y., Mizukami, Y., Sogawa, K., & Fujii-Kuriyama, Y. (1985) J. Biol. Chem. 260, 7980-7984] yet exhibit an intriguing difference in enzymatic activity. Studies to establish the basis for this difference, including a development of the technique of surface-enhanced resonance Raman spectroscopy (SERRS), are reported. Studies on substrate binding, metabolism, and redox properties, as well as SERRS, indicate a significant difference in the heme environment of these two proteins. No significant difference in the interaction of the two proteins with P-450 reductase could be established. However, this interaction appeared sensitive to changes in ionic strength, suggesting ionic interactions are important in the functional coupling of these electron-transport components. A marked variation in the ratio of PB3a to PB3b activity in the metabolism of different substrates, which included a series of structurally similar resorufin analogues, provided further evidence that reductase coupling was not a critical factor. Therefore, the few amino acid differences observed between these proteins indicate sites that may be important in influencing the heme environment of these cytochrome P-450's.

The properties of five structurally related forms of cytochrome P-450 (PB1a, PB1b, PB2a, PB2b and PB2d) isolated from rats treated with phenobarbital have been compared with two forms isolated previously now termed ‘PB1c’ and ‘PB2c’ These enzymes were characterized by their marginal inducibility by phenobarbital and are clearly distinguishable from the major phenobarbital-inducible proteins. PB1a and PB1b differed in Mr (52,700 and 52,900), absorption spectra and papain-proteolysis fragments. However, they had identical N-terminal sequences. PB2a, PB2b and PB2d had apparent Mr values of 52,900, 52,900 and 50,800. PB2a and PB2b had different N-terminal sequences and, after digestion with papain, gave different papain-proteolysis fragments. The N-terminal sequence of PB2b was similar to, but not identical with, that of pregnenolone-16 alpha-carbonitrile-inducible P-450 species, and PB2b was the protein most closely related to PB2c. The extent of immunocross-reactivity among the forms was stronger within, than between, the PB1 and PB2 groups. Even structurally similar forms were functionally diverse, exhibiting large differences in metabolic specificity in the dealkylation of a series of alkoxyresorufins.

Antibodies to four rat liver forms of cytochrome P-450, two phenobarbital-inducible (PB1 and PB2) and two 3-methylcholanthrene-inducible (MC1 and MC2) proteins, have been used to make a structural and functional comparison of rat and human cytochromes P-450. Proteins from both species were identified on Western blots by their reaction with these antibodies. In the human liver preparations, structurally related proteins to PB1 and to PB2 were identified in all the samples tested with apparent Mr values of 51 800 and 54 800 for PB1 and 53 600 and 57 200 for PB2. Considerable variation in the content of the lower-Mr proteins was measured between samples and, as with the rat enzymes, samples which reacted well with anti-PB1 also reacted with anti-PB2, indicating that these proteins are regulated at least to some degree, co-ordinately. The apparent Mr values of the major human proteins identified with anti-MC1 and anti-MC2 were 54 400 and 57 000 respectively. Only six (of 31) human samples contained significant amounts of these proteins. The same six samples which reacted with anti-MC1 also reacted with anti-MC2, again indicating co-ordinate regulation of these two proteins. Antibody inhibition of microsomal 7-ethoxycoumarin and 7-ethoxyresorufin metabolism demonstrated a degree of conservation of substrate specificity related to specific P-450 isoenzymes between the species. However, the contributions of the different P-450 isoenzymes to the human microsomal activity were not always related to the rat enzyme with the highest activity towards these substrates.

Kinetic evaluation of the capacity of human blood serum to form complexes with bovine trypsin generated partition profiles that may be approximated by a series of four intersecting straight lines. Such profiles are suggested to reflect the binding of trypsin to alpha 2-macroglobulin in a kinetically preferred mode (alpha-sites), followed by a subsidiary mode (beta-sites) and finally to alpha 1-antitrypsin. The form of the profile, in addition to revealing a hitherto unreported proteinase-binding capability of alpha 2-macroglobulin (beta-sites), also indicates that saturation of alpha-sites corresponds to a molar binding ratio of alpha 2-macroglobulin/trypsin of 1:2. Finally the profile provides, for certain pathological states, a clinically valuable characteristic.

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.