Characterization of human liver cytochromes P450 by combining the biochemical and proteomic approaches.

Highly purified human liver microsomes were processed by a combination of the biochemical and proteomic methods. Microsomes were purified from the morphologically normal liver tissue obtained from the resected and discarded masses of surrounding liver upon surgical treatment for hemangioma (control) or hepatic metastases arising from colon cancer (pathology). Proteins of each sample were separated by two-dimensional (2-DE) and one-dimensional electrophoresis (1-DE); selected gel regions were excised, in-gel digested and analyzed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Analysis of collected fingerprints has revealed a total of 13 microsomal membrane proteins involved in the biotransformation of xenobiotics. These were disulfide isomerase, flavine monooxygenase, NADPH-cytochrome P450 reductase and 10 cytochrome P450 forms, namely: CYPs 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. These same samples were characterized by the enzymatic assays using the marker substrates for CYPs 1A, 2B, 3A4, 2C and 2E1. Correlations between mass spectrometric data and enzymatic activities were investigated to demonstrate the manner in which the functional and structural aspects of proteomics meet each other in the field of cytochromes P450.

Heme and apoprotein modification of cytochrome P450 2B4 during its oxidative inactivation in monooxygenase reconstituted system.

The mechanism of the cytochrome P450 2B4 modification by hydrogen peroxide (H2O2) formed as a result of partial coupling of NADPH-dependent monooxygenase reactions has been studied in the monooxygenase system reconstituted from the highly purified microsomal proteins: cytochrome P450 2B4 (P450) and NADPH-cytochrome P450 reductase in the presence of detergent Emulgen 913. It was found, that H2O2-mediated P450 self-inactivation during benzphetamine oxidation is accompanied by heme degradation and apoenzyme modification. The P450 heme modification involves the heme release from the enzyme under the action of H2O2 formed within P450s active center via the peroxycomplex decay. Additionally, the heme lost is destroyed by H2O2 localized outside of enzyme's active center. The modification of P450 apoenzyme includes protein aggregation that may be due to the change in the physico-chemical properties of the inactivated enzyme. The modified P450 changes the surface charge that is confirmed by the increasing retention time on the DEAE column. Oxidation of amino acid residues (at least cysteine) may lead to the alteration into the protein hydrophobicity. The appearance of the additional ionic and hydrophobic attractions may lead to the increase of the protein aggregation. Hydrogen peroxide can initiate formation of crosslinked P450 dimers, trimers, and even polymers, but the main role in this process plays nonspecific radical reactions. Evidence for the involvement of hydroxyl radical into the P450 crosslinking is carbonyl groups formation.

Interactions of cytochrome P450 2B4 with NADPH-cytochrome P450 reductase studied by fluorescent probe.

A new method for monitoring the formation of the cytochrome P450 complexes with NADPH-cytochrome P450 reductase (NCPR) is introduced. The method is based on the quenching of fluorescence of NCPR labelled with 7-ethylamino-3-(4'-maleimidilphenyl)-4-methylcoumarin maleimide (CPM). In a monomerized soluble reconstituted system in the absence of phospholipid, cytochrome P450 2B4 and NCPRcpm were shown to form 1:1 complexes with a Kd of 0.038 microM. Formation of the complex follows the kinetics of reversible second order transition with k(on) = 6.5 10(5) M-1 s-1. Application of high hydrostatic pressure induces dissociation of the complex (delta V degrees = -65 mL/mol). Succinylation of the hemoprotein increases the value of Kd to 0.5 microM primarily by decreasing k(on). In contrast to what was shown for intact 2B4, rising pressure does not take apart succinylated hemoprotein and NCPRcpm molecules, but causes some internal transition in their complex that diminishes the quenching. This transition is characterised by a very large volume change (delta V degrees = -155 mL/mol). The following conclusions were drawn: 1) a molecule of 2B4 contains two distinct contact regions involved in the interactions with NCPR. Only one of these regions is polar and highly hydrated in unbound hemoprotein; 2) interactions of the polar regions of 2B4 and NCPR are necessary to bring CPM-labelled cysteine of NCPR in short distance of the heme of 2B4; and 3) some of the lysine residues located in the proximity of the polar binding regions are apparently involved in the formation of the internal salt bridges in the molecule of 2B4.

[Characteristics of peptide preparations obtained during enzymatic hydrolysis and ultrafiltration of milk proteins for use in specialized nutrition products]. / Kharakteristika peptidnykh preparatov, poluchennykh pri fermentativnom gidrolize i ul'trafil'tratsii belkov moloka, prednaznachennykh dlia ispol'zovaniia v spetsializirovannykh produktakh pitaniia.

+Ultrafiltrated bovine milk proteins with whey protein/casein ratios 20/80, 60/40 and 97/3 were subjected to pancreatin hydrolysis, and the hydrolysates obtained were fractionated to high- and low-molecular weight (LMW) fractions by means of ultrafiltration. The preparations obtained were characterized according to routine physico-chemical indices, amino-nitrogen content, amino acids score, molecular weight distribution, and concentration of precipitating milk antigens. The capacity of LMW fractions for inducing oral anaphylactic sensitization in guinea pig and for changing its susceptibility to histamine LD50 was studied. The conclusion has been made that LMW preparations with whey/casein ratios 20/80 and 60/40 could be effectively used in specialized formulae intended for nutrition of allergic children and adults, nursing women, and also for enteral nutrition.