Effects of reduction and ligation of heme iron on the thermal stability of heme-hemopexin complexes.

Hemopexin has two homologous domains (N- and C-terminal domains), binds 1 mole of heme per mole with high affinity (Kd < 1 pM) in a low-spin bis-histidyl complex, and acts as a transporter for the heme. Transport is accomplished via endocytosis without degradation of the protein. Factors that affect stability of the heme coordination complex and potentially heme release in vivo were examined. The effects of temperature on hemopexin, its N-terminal domain, and their respective ferri-, ferro-, and CO-ferro-heme complexes were studied using absorbance and circular dichroism (CD) spectroscopy. As monitored with second-derivative absorbance spectra, the higher order structure of apo-hemopexin unfolds with a Tm of 52 degrees C in 50 mM sodium phosphate buffer and is stabilized by 150 mM NaCl (Tm 63 degrees C). Bis-histidyl heme coordination by hemopexin, observed by Soret absorbance, is substantially weakened by reduction of ferri-heme-hemopexin (Tm 55.5 degrees C) to the ferro-heme form (Tm 48 degrees C), and NaCl stabilizes both complexes by 10-15 degrees C. CO binding to ferroheme-hemopexin restores complex stability (Tm 67 degrees C). Upon cooling, unfolded apo- and ferriheme-hemopexin extensively refold and recover substantial heme-binding activity, but the characteristic ellipticity of the native protein (UV region) and heme complex (Soret region) are not regained, indicating that altered refolded forms are produced. Lowering the pH from 7.4 to 6.5 has little effect on the stability of the apo-protein but increases the Tm of heme complexes by 5-12 degrees C. The stability of the apo-N-terminal domain (Tm 53 degrees C) is similar to that of intact hemopexin, and the ferri-, ferro-, and CO-ferro-heme complexes of the N-terminal domain have Tm values of 53 degrees C, 33 degrees C, and 75 degrees C, respectively.

[The immunochemical mechanisms of body adaptation to a chemical environment]. / Immunokhimicheskie mekhanizmy adaptatsii organizma k okruzhaiushchei khimicheskoi srede.

For the first time the dynamics of metabolic and immune responses to xenobiotics were analyzed in their long-term effect on the organism. It was shown that the organism gradually lost the capacity to respond to xenobiotics with the induction of hepatic cytochrome P450 and immune response. Possible mechanisms of this phenomenon are under consideration.

[Cytochrome P-450 induction and the subsequent induction of an immune response in rats during the chronic administration of xenobiotics]. / Induktsiia tsitokhroma P-450 i posleduiushchaia induktsiia immunnogo otveta u krys pri khronicheskom vvedenii ksenobiotikov.

The long-term administration of xenobiotics carcinogens o-aminoazotoluene (o-AAT) and benz(a)pyrene (BP) to rats was found to cause induction of the liver cytochrome P-450 system which gradually decreases in spite of continued administration of the agents. Induction of microsomal oxygenases under these conditions is followed by induction of the immune response to o-AAT and BP. The data obtained correspond to the conception of the immunochemical functional system of homeostasis implying that the cytochrome-450 system and the immunity system are functionally linked and are elements of the common functional adaptive system of the organism.

[Immune response to benzo(a)pyrene in rabbits immunized with a conjugate of benzo(a)pyrene-albumin, synthesized in the microsomal monooxygenase system of the liver]. / Imunnyi otvet na benzo(A)piren u krolikov, immunizirovannykh kon''iugatom benzo(A)piren-al'bumin, sintezirovannym v mikrosomalnoi monooksigenaznoi sisteme pecheni.

The feasibility of obtaining a conjugated benz(a)pyrene-protein antigen in the liver cytochrome P-450 system was studied. Covalent binding of benz(a)pyrene (BP) to albumin was performed with the use of liver microsomal fractions of 3-methylcholanthrene-induced rabbits. It was demonstrated that BP oxidation in liver microsomes is accompanied by covalent binding of [14C]BP to exogenous rabbit albumin. Immunization of rabbits with the obtained conjugate results in the development of a specific immune response to BP, and the appearance of specific antibodies and lymphocytes specifically binding [14C]BP in the blood.

[Cytochrome P-450-dependent mechanisms of the biosynthesis of protein-conjugated benzo(a)pyrene antigens and their role in the development of a specific immune response to this carcinogen]. / Izuchenie tsitokhrom-P-450-zavisimykh mekhanizmakh biosinteza kon''iugirovannykh antigenov benzo(a)pirena s belkami i ikh rol' v razvitii spetsificheskogo immunnogo otveta na étot kantserogen.

There was studied the possibility of covalent binding to proteins of a carcinogenic compound benzo(a)pyrene in the system of cytochrome P-450 of the liver and the possibility of the development of the immune reaction to administration of the conjugated antigens obtained in such a way to animals. It was shown that under experimental conditions modelling the processes of microsomal oxidation of benzo(a)pyrene in the organism there occurs irreversible binding of 14C-benzo(a)pyrene both to microsomes and the added albumin. Immunization of rabbits by conjugates benzo(a)pyrene-albumin and benzo(a)pyrene-microsomes leads to the development of the immune response to the original carcinogen. The antibodies and lymphocytes specifically binding 14C-benzo(a)pyrene appear in the blood.

[Reciprocal effect of cyclophosphane on the liver cytochrome P-450 and immunity system]. / Retsiproknoe vliianie tsiklofosfana na tsitokhrom P-450 pecheni i sistemu immuniteta.

It has been demonstrated in experiments on mice that administration of cyclophosphamide in a dose of 50 mg/kg for 3 days provokes marked induction of liver cytochrome P-450. However, 5 days after completing cyclophosphamide administration the induction is replaced by inhibition. A similar action under analogous conditions is produced by 3-methylcholanthrene which is widely used as an inductor of cytochrome P-450. The induction of liver cytochrome P-450 by cyclophosphamide or 3-methylcholanthrene is accompanied by pronounced inhibition of immunoreactivity. The known inductor phenobarbital does not produce any reciprocal action. Apparently, the reciprocal action of cyclophosphamide and 3-methylcholanthrene on cytochrome P-450 and immunity is a consequence of intense formation with the involvement of cytochrome P-450 of highly reactive metabolites interfering with the function of immunocompetent cells.