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Using electrospray ionization tandem mass spectrometry, a comparative analysis of the HaCaT keratinocyte proteins encoded by the 18th chromosome was performed before and after exposure to sodium dodecyl sulfate (25 mg/ml) and to Triton X-100 (12.5 mg/ml) in a subtoxic dose for 48 hours. Proteins were identified using the SearchGUI platform (X!Tandem and MS-GF+ search engines). In total, 1284 proteins were found in immortalized human HaCaT keratinocytes and about 75% of them were identified by two or more peptides. Were identified, that 26 proteins were encoded by genes of chromosome 18. Among these proteins, 17 were common for control cells and HaCaT cells treated with SDS. Proteins MARE2 and CTIF were identified only in control keratinocytes. Seven identified proteins encoded by genes of chromosome 18 were found only in detergent-treated keratinocytes: LMAN1, NDUV2, SPB3, VPS4B, KDSR, ROCK1 and RHG28.Metodom tandemnoĭ mass-spektrometrii s élektrospreĭnoĭ ionizatsieĭ proveden sravnitel'nyĭ analiz belkov keratinotsitov linii NaSaT, kodiruemykh genami 18 khromosomy, do i posle vozdeĭstviia subtoksicheskikh doz dodetsilsul'fata natriia (DSN; 25 mkg/ml) i tritona X-100 (12,5 mg/ml) v techenie 48 ch. Identifikatsiiu belkov provodili s ispol'zovaniem platformy SearchGUI (poiskovye sistemy X!Tandem i MS-GF+). Vsego v immortalizovannykh keratinotsitakh cheloveka linii NaSaT obnaruzheno 1284 belka, iz nikh okolo 75% identifitsirovano po dvum i bolee peptidam. Dostoverno identifitsirovany 26 belkov, kodiruemykh genami 18 khromosomy; iz nikh 17 iavliaiutsia obshchimi dlia kontrol'nykh kletok i kletok NaSaT posle vozdeĭstviia DSN i 15 — obshchimi dlia kontrol'nykh kletok i kletok, obrabotannykh tritonom X-100. Belki MARE2 i CTIF identifitsirovany tol'ko v kontrol'nykh keratinotsitakh. Sem' identifitsirovannykh belkov, kodiruemykh genami 18 khromosomy, opredeleny tol'ko v obrabotannykh DSN keratinotsitakh, LMAN1, NDUV2, SPB3, VPS4B, KDSR, ROCK1, RHG28.

The relative differences between post-translational modifications (PTM) of proteins in blood plasma samples of patients with cerebral ischemia (CI) and healthy people were investigated using of the method of label-free comparative proteomic analysis based on the technology of tandem HPLC-MS/MS. For PTM detection we used multiple MS/MS search in the database Mascot for variable PTM and Progenesis LS-MS software. In the CI plasma samples, we observed an increase in the proportion of peptides with such PTM as phosphorylation of serine, threonine, and tyrosine, acetylation of lysine and protein N-term, ubiquitination of lysine and deamidation of glutamine related to clinically significant processes were revealed.Issledovany otnositel'nye razlichiia posttransliatsionnykh modifikatsiĭ (PTM) belkov v obraztsakh plazmy krovi bol'nykh tserebral'noĭ ishemieĭ (TsI) i zdorovykh liudeĭ na osnove metoda sravnitel'nogo bezmetkovogo proteomnogo analiza na baze tekhnologii tandemnoĭ VÉZhKh-MS/MS. Dlia obnaruzheniia PTM byl vypolnen mnozhestvennyĭ poisk MS/MS v baze dannykh Mascot s ustanovlennoĭ peremennoĭ PTM s ispol'zovaniem programmnogo obespecheniia Progenesis LS-MS. V plazme bol'nykh TsI nabliudali uvelichenie doli posttransliatsionno-modifitsirovannykh peptidov. Vyiavleny takie PTM belkov, kak fosforilirovanie ostatkov serina, treonina i tirozina, atsetilirovanie ostatkov lizina i N-kontsevoe atsetilirovanie, ubikvitinirovanie ostatkov lizina i dezamidirovanie ostatkov glutamina i asparagina, imeiushchie otnoshenie k klinicheski znachimym protsessam.

In the present study, we explored the technology of liquid chromatography-mass spectrometry (HPLC-MS/MS) for the proteome analysis of blood plasma of patients with early chronic cerebral ischemia. Analysis of mass-spectrometer data carried out in automatic mode using the software Progenesis LS-MS. As a result of this study identified 43 proteins. The differences identified in the study group compared with the control in 7 proteins. It was found that in the early stages of chronic cerebral ischemia proteome changes in blood plasma affect proteins related to the immune system, the system for the maintenance of hemostasis and lipid metabolism.Metodom sravnitel'nogo proteomnogo analiza na baze tekhnologii tandemnoĭ zhidkostnoĭ khromato-mass- spektrometrii VÉZhKh-MS/MS issledovali belkovyĭ profil' plazmy krovi bol'nykh khronicheskoĭ tserebral'noĭ ishemieĭ. Analiz mass-spektrometricheskikh dannykh, provedennyĭ v avtomatizirovannom rezhime s ispol'zovaniem programmnogo obespecheniia Progenesis LS-MS, pozvolil identifitsirovat' 43 belka. Vyiavleny razlichiia v issleduemoĭ gruppe po sravneniiu s kontrolem po 7 belkam, otnosiashchimsia k immunnoĭ sisteme, sisteme podderzhaniia gemostaza i metabolizma lipidov.

Blood plasma proteome in patients with cerebral ischemia and healthy individuals was studied using comparative proteomic analysis based on tandem HPLC-MS/MS. Mass spectra were analysed in an automated mode using Progenesis LS-MS software and 256 proteins were identified. Significant quantitative differences were revealed for 20 proteins. It was found that changes in the blood plasma proteome in subjects with cerebral ischemia involved a wide range of proteins: molecular chaperones, fibrinolysis, angiogenesis, and immune system proteins, proteins involved in homeostasis maintenance, cell differentiation and proliferation, regulators of apoptosis, and cytoskeleton proteins.

The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.

The effects of phosphatidylcholine-based phospholipid nanoparticles containing fullerene C60 on Danio rerio fish embryos were studied. Exposure of the embryos with the nanoparticles for 48 h did not lead to appreciable changes in the number of protein bands in SDS-PAGE in comparison with the control (exposure in medium with phosphatidylcholine). Mass spectrometric identification of proteins showed differences in the proteomic profiles of the samples. The content of vitellogenins changed after exposure with phosphatidylcholine-based nanoparticles with C60 fullerenes. This could indicate low toxicity of the nanoparticles towards D. rerio embryos under experimental conditions.

This study was designed to track changes in the blood plasma proteome caused by exposure of healthy humans to dry immersion. The blood plasma specimens of five healthy volunteers aged 23–29 years obtained seven days before immersion, on day 7 of exposure in the immersion bath, and seven days after completion of the experiment were analyzed. After the extraction of major and the concentration of minor proteins, depleted blood plasma fractions were subjected to two-dimensional electrophoresis with the subsequent identification of significantly different protein spots with the method of mass-spectrometric analysis of peptide fragments. A significant change in intensity was observed for 25 protein spots with a coefficient of variation of >60%. Cluster analysis confirmed substantial changes in the blood plasma proteome in the period of readaptation of the body to the conditions of normal vital activity upon completion of immersion. On day 7 of readaptation, the apolipoprotein A-1, A-IV, and E concentrations were significantly higher compared to the baseline data and the measurements on day 7 of immersion. However, the levels of α-, β-fibrinogen, the complement C4B factor, and serum amyloid P were found to be lower.

Using one-dimensional proteomic mapping (combination of one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry MALDI-TOF-PMF) the protein profile of Danio rerio embryos has been investigated. The fish species Danio rerio is the most effective alternative model of vertebrates used for studies of drug toxicity (e.g. doxorubicin) due to its high degree of homology with human genome. The proteomic profiling resulted in identification of 84 proteins, including 15 vitellogenins. Using the procedure of preparation of homogenates of Danio rerio embryos optimized by ultrasonic treatment promoting removal of yolk basic proteins (vitellogenin) we have registered changes in the proteome profile of D. rerio embryos induced by doxorubicin (DOX). Growth D. rerio embryos in the medium with DOX caused the decrease in the number of vitellogenins, disappearance of cardiac troponins, and induction of caspase-3. All these observations are consistent with the literature data on doxorubicin-induced cardiotoxicity. The proposed method of 1D proteomic mapping may be used not only for protein identification but also for registration of changes in embryonic proteomic profile caused by drugs or any toxic compound for studying the mechanisms underlying induced toxicity.

In the present study, a proteomic technology combining one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry (MALDI-TOF-PMF) has been successfully applied for revelation of changes in the protein profile of zebrafish (Danio rerio) 52 hpf embryos. Prior to 1DE separation of zebrafish embryonic proteins, the procedure for obtaining embryos homogenate was optimized by ultrasonic treatment. A total of 84 proteins, including 15 vitellogenins, were identified. It was shown that growing of zebrafish embryos in the medium with doxorubicin (DOX) stimulated Caspase-3 induction and promoted the disappearance of cardiac troponins, both these findings being consistent with literature data on doxorubicin-induced cardiotoxicity. The 1DE-based proteomic mapping approach proposed herein enabled not only to identify proteins but also to register those changes in embryos' proteomic profile that were caused by doxorubicin.

Cytochromes P450 are the key enzymes for activating and inactivating many drugs; individual expression levels of CYPs may play a crucial role in drug safety and drug efficacy. Statistical comparison of biochemical profiles of 23 human liver microsomes have been used to characterize human liver samples. The profile included 12 parameters, namely activity of NADPH-cytochrome P450 reductase, cytochrome P450 content and cytochrome P450-dependent monooxygenase activities with marker substrates. Unsupervised statistical methods including cluster analysis and principal component analysis revealed with very high confidence the presence of two groups. Difference between the groups was explained by peculiarities of reductase activity and cytochrome P450 enzyme activities with 7-ethoxyresorufin, 7-methoxyresorufin, 7-methoxycoumarin, 7-benzyloxyresorufin and 7-benzyloxyquinoline. Results of biochemical assays coupled with multidimensional data analysis can be further used for targeted proteomic profiling of microsome oxidation mechanisms.

The current investigation was undertaken with the aim to carry out an in vitro evaluation of the ability of coumarin derivatives as probe substrates to predict the activity of CYP51b1. The results obtained indicate that 7-aminocoumarin-4-acetic acid (ACAC) can be used to determine the recombinant CYP51b1 activity. Determination of CYP51b1 activity with ACAC is based on the direct registration of fluorescence increasing at 30°C. It was found also that BMR in a simple soluble model system can be used as an electron donor for CYP51B1. Fluorescence-based assay is highly sensitive and can be used for the screening of sterol 14alpha-demethylase inhibitors.

Direct electron transfer has been demonstrated between cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14α-demethylase (CYP51MT) and screen printed graphite electrodes, modified by gold nanoparticles and didodecyldimethyl ammonium bromide (DDAB). The proposed method for preparation of enzymatic nanostructured electrodes may be used for electrodetection of this hemoprotein provided that 2–200 pmol P450 per electrode has been adsorbed. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, lanosterol) and inhibitor ketoconazole were analyzed using cyclic voltammetry (CV), square wave (SWV) or differential pulse (DPV) voltammetry, and amperometry.

The electrochemical reduction of the heme protein sterol-14alpha-demethylase from Mycobacterium tuberculosis (CYP51b1, or further CYP51) was investigated. Direct electron transfer was demonstrated between CYP51 and graphite screen-printed electrodes modified with gold nanoparticles and with the membrane-like synthetic surfactant didodecyl dimethylammonium bromide. The formal potential of the Fe3+/Fe2+ pair, E(1/2), is equal to -273 mV (vs. Ag/AgCl). The cathodic current corresponding to the reduction of oxygen by immobilized heme protein was registered in the presence of oxygen. Addition of lanosterol, one of the substrates of the CYP51 family, to the oxygenated solution caused a concentration-dependent increase in the reduction current in voltammetric and amperometric experiments. Ketoconazole, an inhibitor of CYP51, inhibited the catalytic cathodic current in the presence of lanosterol. Electrochemical reduction of CYP51 may serve as an adequate alternative to the reconstituted system, which requires additional redox partners for the exhibition of catalytic activity of heme proteins of the cytochrome P450 superfamily.

The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.Issledovali belkovyĭ profil' émbrionov ryb vida Danio rerio, vyrashchennykh v srede s doksorubitsinom, vkliuchennym v fosfolipidnuiu transportnuiu nanosistemu (doksolip), ispol'zuia sochetanie odnomernogo élektroforeza s posleduiushcheĭ vremiaproletnoĭ mass-spektrometrieĭ s lazernoĭ desorbtsieĭ i ionizatsieĭ (MALDI-TOF-PMF). Pri vyrashchivanii émbrionov D. rerio v srede s doksolipom otmecheno sushchestvennoe uvelichenie kolichestva tsitoskeletnykh belkov, snizhenie kolichestva iadernykh belkov, uchastvuiushchikh v sinteze DNK i RNK, i ischeznovenie vitellogenina 2 po sravneniiu s kontrolem (sreda s fosfolipidnoĭ transportnoĭ nanosistemoĭ). Analiz proteomnykh profileĭ émbrionov, podvergnutykh obrabotke doksolipom, svidetel'stvuet o snizhenii émbriotoksichnosti doksorubitsina pri vkliuchenii ego v fosfolipidnuiu nanosistemu.

We identified changes in the proteome of healthy human blood plasma caused by exposure to 105-day confinement in an isolation chamber. After removal of major proteins and concentration of minor proteins, plasma fractions were analyzed by two-dimensional electrophoresis followed by identification of significantly different protein spots by mass spectrometric analysis of the peptide fragments. The levels of α- and β-chains of fibrinogen, a fragment of complement factor C4, apolipoproteins AI and E, plasminogen factor C1 complement, and immunoglobulin M changed in participants during the isolation period. These changes probably reflect the adaptive response to altered conditions of life.

Carbon monoxide (CO) formation was studied in the process of lipid peroxidation in phenobarbital-induced rabbit liver microsomes. The reaction was NADPH-dependent and required Fe(2+), which occurs in microsomes as being protein bound and is not a consequence of heme destruction. Zn-protoporphyrin IX, an inhibitor of the heme oxygenase activity, proved to have no effect on CO production, suggesting that heme oxygenase is not involved into the CO generation reaction. At the same time, the addition of cytochrome P450 typical inhibitors SKF 525A and metyrapone to the reaction mixture had an inhibitory effect on the CO formation rate. Antioxidants such as alpha-tocopherol and desferal inhibited lipid peroxidation in phenobarbital-induced rabbit liver microsomes, and in this case the CO production was not registered. Thus, on the basis of the results presented here it is possible to assert that the process of NADPH, Fe(2+)-dependent carbon monoxide formation in microsomes is a result of lipid peroxidation with cytochrome P450 2B4 participation.

Background Vitellogenin (Vtg) is the major egg yolk protein (YP) in most oviparous species and may be useful as an indicator in ecotoxicological testing at the biochemical level. In this study, we obtained detailed information about the Vtgs of Danio rerio embryos by cutting SDS-PAGE gel lanes into thin slices, and analyzing them slice-by-slice with (MALDI-TOF) mass spectrometry. Results We conducted three proteomic analyses, comparing embryonic Danio rerio Vtg cleavage products after exposure for 48 h to CdSecore/ZnSshell quantum dots (QDs), after exposure to a mixture of the components used for quantum dot synthesis (MCS-QDs), and in untreated embryos. The Vtg mass spectrometric profiles of the QDs-treated embryos differed from those of the unexposed or MCS-QDs-treated embryos. Conclusion This study demonstrates the possible utility of Vtg profiling in D. rerio embryos as a sensitive diagnostic tool to estimate nanoparticle toxicity. Electronic supplementary material The online version of this article (doi:10.1186/s12953-015-0072-7) contains supplementary material, which is available to authorized users.

BackgroundThere are two ways that statistical methods can learn from biomedical data. One way is to learn classifiers to identify diseases and to predict outcomes using the training dataset with established diagnosis for each sample. When the training dataset is not available the task can be to mine for presence of meaningful groups (clusters) of samples and to explore underlying data structure (unsupervised learning).ResultsWe investigated the proteomic profiles of the cytosolic fraction of human liver samples using two-dimensional electrophoresis (2DE). Samples were resected upon surgical treatment of hepatic metastases in colorectal cancer. Unsupervised hierarchical clustering of 2DE gel images (n = 18) revealed a pair of clusters, containing 11 and 7 samples. Previously we used the same specimens to measure biochemical profiles based on cytochrome P450-dependent enzymatic activities and also found that samples were clearly divided into two well-separated groups by cluster analysis. It turned out that groups by enzyme activity almost perfectly match to the groups identified from proteomic data. Of the 271 reproducible spots on our 2DE gels, we selected 15 to distinguish the human liver cytosolic clusters. Using MALDI-TOF peptide mass fingerprinting, we identified 12 proteins for the selected spots, including known cancer-associated species.Conclusions/significanceOur results highlight the importance of hierarchical cluster analysis of proteomic data, and showed concordance between results of biochemical and proteomic approaches. Grouping of the human liver samples and/or patients into differing clusters may provide insights into possible molecular mechanism of drug metabolism and creates a rationale for personalized treatment.

Formation of binary and ternary complexes in the water-soluble cytochrome P450cam (P450cam)-containing as well as in the membrane P4502B4(2B4)- and the mixed P450scc-containing monooxygenase systems was investigated in real time by the ‘resonant mirror’ optical biosensor method. It was shown that the inter-protein electron transfer occurs not only during complex formation but also upon random collision — as was the case with the d-Fp/d-b5 pair (2B4 system). Binary complexes may be either facilitative to electron transfer (electron-transfer complexes) or prohibitive to it (non-productive complexes). Although the binary PdR/Pd and P450cam/Pd complex formation (within the P450cam-system) as well as the binary AdR/Ad and P450scc/Ad complex formation (within the P450scc-system) does occur, the lifetimes of these complexes formed are several orders of magnitude higher than the time required for realization of a complete hydroxylation cycle. At the same time, the lifetimes of the ternary PdR/Pd/P450cam and AdR/Ad/P450scc complexes are sufficient to permit the realization of a complete hydroxylation cycle in either of these systems. For the membrane P450 2B4 system, the formation of both the binary (Fp/2B4 and 2B4/b5) and ternary (Fp/2B4/b5) complexes was registered. The lifetimes of the binary Fp/2B4 and the ternary Fp/2B4/b5 complexes are sufficient for realization of a complete hydroxylation cycle in each of them.

This paper describes a specialized database dedicated exclusively to the cytochrome P450 superfamily. The system provides the impression of superfamily's nomenclature and describes structure and function of different P450 enzymes. Information on P450-catalyzed reactions, substrate preferences, peculiarities of induction and inhibition is available through the database management system. Also the source genes and appropriate translated proteins can be retrieved together with corresponding literature references. Developed programming solution provides the flexible interface for browsing, searching, grouping and reporting the information. Local version of database manager and required data files are distributed on a compact disk. Besides, there is a network version of the software available on Internet. The network version implies the original mechanism, which is useful for the permanent online extension of the data scope.

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.

A method for direct registration of CYP51b1 (sterol-14α-demethylase) activity with coumarin derivatives as substrate has been proposed. Determination of catalytic activity of this enzyme with 7-aminocoumarin-4-acetic acid (ACAC) is based on registration of the increase of fluorescence (λexcitation = 360 nm and λemission = 435 nm) at 30°C. In the model system for assay of CYP51b1 activity BMR (a flavin domain of the bacterial cytochrome P450BM-3) may serve as the electron donor. The developed method is simple, highly sensitive and accurate; it can be used for screening of sterol 14α-demethylase inhibitors.

Proteomics often exploits sequential cutting of the SDS-PAGE gel lane into slices with subsequent identification of proteins by peptide mass fingerprinting (PMF). In this paper, the influence of slice thickness on protein identification was investigated. Following the separation of the human liver microsomal fraction, the 37 to 75 kDa range of the gel lane was cut into 20 or 40 slices of 0.5 mm and 0.25 mm thickness, respectively. Identification of proteins after trypsinolysis was performed by MALDI-TOF mass spectrometry. A twofold reduction of slice thickness did not influence the number of peaks in PMF-spectra. It was established that at a tolerance of 0.15 Da the number of identified proteins in the 40 slices series exceeded by more than twofold the number of identifications in 20 slices series. It was shown that the decrease of slice thickness leads to a considerable enhancement of peptide peak’s intensity and, also, to the changes of PMF-spectra which resulted gaining additional peptides for enrichment of sequence coverage.

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H 2 O 2 formed through peroxy complex breakdown, whereas H 2 O 2 formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H 2 O 2 produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b 5 , NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H 2 O 2 is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.

The current investigation was undertaken with the aim to carry out an in vitro evaluation of the ability of coumarin derivatives as probe substrates to predict the activity of CYP51b1. The results obtained indicate that 7-aminocoumarin-4-acetic acid (ACAC) can be used to determine the recombinant CYP51b1 activity. Determination of CYP51b1 activity with ACAC is based on the direct registration of fluorescence increasing at 30 degrees C. It was found also that BMR in a simple soluble model system can be used as an electron donor for CYP51B1. Fluorescence-based assay is highly sensitive and can be used for the screening of sterol 14alpha-demethylase inhibitors.

Proteomic approaches have been used for detection and identification of cytochromes P450 forms from highly purified membrane preparations of human liver. These included the protein separation by 2D-and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in a range 45–66 kDa (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of an original 1D-ZOOMER software package which allowed to carry out the processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range 45–66 kDa we identified 13 microsomal membrane proteins including such cytochrome P450 forms as CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. Study of enzymatic activities of human liver microsomal cytochrome P450 isoforms CYP 1A, 2B, 3A, and 2E revealed the decrease in the rates of O-dealkylation and N-demethylation catalyzed by CYP 450 1A1/1A2 and 3A4 under pathological conditions, whereas 7-benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP 2B and CYP 2C), the activities of CYP 2E1 (methanol oxidation), 7-pentoxyresorufin-O-dealkylation (CYP 2B), 7-ethoxy-and 7-methoxycoumarin-O-dealkylases (CYP 2B1) remained basically unchanged.

A method for constructing one-dimensional proteomic maps (1D-PM) based on mass spectrometric identification of proteins from adjacent slices of one-dimensional electrophoregram has been developed. For the proteomic mapping, gel lanes were sectioned into slices less than 0.2 mm thick and each slice was subjected to enzymatic hydrolysis. The resultant mixture of peptide fragments was analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteins were identified by the mass spectra obtained. Data on peptide fragments and corresponding identified proteins were presented as a 1D-PM. Proteomic maps were constructed by assigning individual proteins to gel slices based on number of matching peptides in a corresponding MS-data. On 1D-PM of human liver microsomal fraction, 18 proteins were identified in the region of 40–65 kDa. These included 12 membrane proteins belonging to the superfamily of cytochromes P450. Pooling of mass spectrometric data, obtained from several adjacent gel slices (molecular zooming) increased sequence coverage of CYP2A (cytochrome P450 family 2A). The maximal coverage of 66% significantly exceeded the level of 48% that could be obtained using one (even the most informative) slice. This method can be applied to the proteomic profiling of membrane-bound proteins.

In part 2 of the review the authors consider factors, having influence on the state of monooxygenase system (MOS): cytochrome P-450 gene polymorphism, induction or inhibition of these systems under effect of drugs, crossed substance specificity of cytochrome P-450 forms. Various methodical approaches (genomic, proteomic, bioelectrical technologies, therapeutic drug monitoring) to receive full information about a profile of cytochrome P-450 for every specific person, are compared. Necessity of MOS individual features assessment for optimization of drug therapy is proved.

The review summarizes data on the molecular basis of medication metabolism, factors forming the personal profile of cytochrome P-450, methods to estimate the activity of the monooxygenase system, and problems of medication interference and therapeutic pharmacomonitoring, forming the basis of personified medicine. Special attention is paid to the cytochrome-P-450-containing hepatic system, responsible for inter-individual differences after administration of medications in therapeutic doses. Factors influencing the condition of the monooxygenase system: cytochrome P-450 genetic polymorphism, inducing of inhibition of these systems by medications, and crisscross substrate specificity of cytochrome P-450 are considered. Different methodical approaches (genomic and proteomic ones, bioelectrochemical nanotechnologies, and therapeutic pharmacomonitoring) to obtaining full information on cytochrome P-450 for each individual are compared. The article substantiates the necessity to assess individual features of the monooxygenase system to optimize medication therapy.

Proteomic approaches have been used for detection and identification of cytochromes P450 from highly-purified membrane preparations of human liver. These included the protein separation by 2D- and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in the range of 45-66 kD (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of original 1D-ZOOMER software package which allowed to carry out processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range of 45-66 kDa we identified 13 microsomal membrane proteins including 11 cytochromes P450, namely CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. The microsomal samples were characterized by the enzymatic assays using the marker substrates for CYP1A, 2B, 3A4, 2C and 2E1. The 7-methoxy- and 7-ethoxyresorufin-O-dealkylase activities (i.e. the marker activities for cytochromes P450 1A1/1A2, respectively) and the erythromycin-N-demethylase activity (i.e. the marker activity for cytochrome P450 3A4) are lowered in pathology compared to these activities in norm. At the same time the benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP2B and CYP2C), the activities of CYP2E1 (methanol), 7-pentoxyresorufin-O-dealkylation (CYP2B), 7-ethoxy- and 7-methoxycoumarin-O-dealkylases (CYP2B1) did not change. On the basis of the results obtained efficiency of a combination proteomic and biochemical analyses for inventory cytochromes P450 and revealing of their level expression is shown, and opportunities of mass spectrometry for a quantitative estimation of proteins are discussed.

The present study demonstrates direct electron transfer between cytochromes P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51b1) on the one hand and screen-printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB) on the other. Electro detection of heme proteins was possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, and lanosterol) and with P450 inhibitor (ketoconazole) were analyzed using cyclic voltammetry (CV), square wave voltammetry (SWV) differential pulse voltammetry (DPV), and amperometry.

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.

CYP51 family of cytochromes P450 (sterol 14-alpha-demethylases) comprises the representatives from different kingdoms of living world, thus positioning itself as the most ancient member of the superfamily. In the course of the present research the collection of 36 full-length CYP51 amino acid sequences was submitted to cluster analysis. Each node of the clustering dendrogram corresponds to the groups of proteins, located on the branches descending from the node. By making the multiple alignment of each group of protein sequences we obtained the node-specific consensus sequences. The informational content of the consensus was defined as the presence of the compact conserved sites, the motifs. The assessment of informational content was computed using Sherman's non-parametric statistical criterion. The high informational content was observed for the 100% conserved consensus sequences of the following CYP51,s groups: fungi, animal+plant, plant+protista and bacteria. These selected consensus sequences were next aligned all together to get the final consensus for the whole family. To enrich the informational content of the CYP51 consensus the level of its conservation was dropped to 75%. Regions of statistically significant conservation were unraveled in the CYP51 consensus sequence. These regions (motifs) were then correlated with the information on secondary structure elements and substrate recognition sites reported for CYP51 from Mycobacterium tuberculosis. Seven motifs appeared to be obligatory for every CYP51 protein. The motifs thus obtained were searched for among all the known cytochrome P450 proteins. Some motifs were found to be absolutely specific for 14-alpha-demethylases, whereas others were common to different species of cytochromes P450.

Proteome maps of microsomes and their ghosts (i.e. membranes purified from "ballast" proteins) were obtained using highly purified mouse liver microsomes. Comparative analysis of protein composition of ghosts without and after the induction with phenobarbital (cytochromes P450 inducer) by using 1D- and 2D-electrophoresis and MALDI-TOF-mass-spectrometry revealed more than 30 new proteins, in the course of induction in the 45-60 kDa range (corresponding to the mol. weights of cytochromes P450). In the 17 kDa range (corresponding to the mol. wt. of cytochrome b5) there were 4 additional protein stains about 20 proteins disappeared over the entire electrophoregram). Separation of microsomal ghosts proteins by 1D electrophoresis followed by mass-spectrometric analysis allowed to identify cytochromes P450. The present investigation demonstrates the efficiency of different proteomic methods combination (1D- and 2D-electrophoresis, mass-spectrometry, bioinformatics and determination of the enzyme activities) for cytochromes P450 identification and elucidation of their functioning in different animal tissues and then extrapolating this approach to humans.

The efficiency of the proteomic approach for the revelation of proteins, including components of the liver microsomal monooxygenase system (cytochromes b5 and P450) was demonstrated. The liver microsomes and their ghosts (i.e. membranes devoid of “ballast” proteins) were prepared from the control and phenobarbital-treated mice. Microsomes and their ghosts were characterized using the conventional biochemical assay and analysed by one- and two-dimensional electrophoresis (1-DE and 2-DE, respectively) coupled with MALDI-TOF peptide mass fingerprinting procedure. Catalytic activity of cytochromes P450 was measured using specific fluorogenic substrates for CYP1A, CYP2A, CYP2B and CYP2C families. The protein composition of control and phenobarbital-induced ghosts was analysed. The proteomic 2D-based protein separation method enabled us to reveal up to 1005 proteins, the majority of them being soluble. Among the 34 identified proteins, the cytochrome b5-like protein was revealed; however, cytochromes P450 appeared to be undetectable under 2-DE separation conditions. The separation of microsomal ghosts proteins by 1-DE, followed by mass-spectrometric analysis of bands from the 45 to 66 kDa gel range made it possible to identify hydrophobic proteins including cytochromes P450 (CYP2A4 and CYP2A5) and dimethylaniline monooxygenase. The high O-deethylation rate of 7-ethoxycoumarin—a substrate for rodent CYPs 2A and 2B, in particular for CYP2A5—was observed, in agreement with the results of mass-spectrometric identification. Collectively, the data obtained indicate that a combination of enzyme activity assays and various protein separation techniques coupled with mass-spectrometric protein identification allows a more comprehensive insight into the machinery of the cellular detoxifying system.

Protective influence of a new phospholipid preparation "Phospholiv" was studied using a model of chronic hepatitis. Animals were treated 45 days intraperitoneay with CCl4 with parallel intragastral administration of Phospholiv or--(for comparison)--the of other phospholipid hepatoprotector, Essential. Morphologic changes of liver, as well as protein and RNA biosynthesis were evaluated in the end of experiment--by means of measuring C14-leucine and C14-orotic acid incorporation into hepatocyte subcellular fractions. Both phospholipid preparations attenuated dystrophic liver changes, Phospholiv effect being more pronounced. They both prevented CCl4 induced inhibition of label incorporation into subcellular fraction proteins, but only Phospholiv, promoted the maintaining normal level of radioactivity incorporation into cytosol proteins and hepatocyte RNA. The results, confirming certain protective effect of Essential, show more pronounced hepatoprotective action of the new preparation Phospholiv (developed on the basis of polyunsaturated phosphatidylcholine and glycyrrhizinic acid salt). Data show also on possible fit hepatitis treatment.

Curative effect of new preparation "Phospholiv", elaborated in Institute of Biomedical Chemistry, in acute CCl4 induced rat hepatit model was studied. The preparation consists of polyunsaturated phosphatidylcholine and glycyrrhizinic acid salt. Recovery of damaged biosynthesis of albumin and total cell liver RNA--by incorporation of C14-leucine and C14-orotic acid--were observed after 3 days Phospholiv administration, that showed on reparation of damaged protein-synthesis system. Label incorporation into liver fraction80S--that was decreased under CCl4 influence--was also restored after Phospholiv treatment, that may testify on its regenerative effect on wholeness of subcellular hepatocytes structures. Substantial decrease of morphologic damages of liver tissue was demonstrated as well. Other phospholipid preparation--known hepatoprotector Essentiale--gave some positive effects too, but Phospholiv influence on biochemical and morphological liver features were 1.5-2 fold as compared with that of Essentiale. Results show on efficiency of polyunsaturated phospholipids in the treatment of acute hepatit in rats--as a result of influence on hepatocytes cell membrane--and on preferential effect of new hepatoprotector Phospholiv.

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.

Possible mechanisms of cytochrome P4502B4 modification by H2O2 formed during catalytic turnover have been studied. Oxidative self-inactivation of cytochrome P4502B4 in monooxygenase system, reconstituted from highly purified membrane proteins: cytochrome P4502B4, NADDPH-cytochrome P450 reductase and cytochrome b5 in the presence of detergent Emulgen 913, involves the heme destruction and apoenzyme modification. The cytochrome P450 self-inactivation is accompanied by protein aggregation, oxidation of SH-groups and changes of the surface charge. H2O2 and non-specific radical reactions may be responsible for the intermolecular cross-linking. Oxidative modification of cytochrome P4502B4 may be initial stage of the protein decay in the cell.

The mechanism of cytochrome P450 2B4 self-inactivation during catalytic turnover has been studied in monooxygenase reconstituted system containing from monomers of the membrane proteins: NADPH-cytochrome P450 reductase, cytochrome P450 2B4 and cytochrome b5 in presence of detergent emulgen 913. It was shown that P450 is inactivated at a high rate during benzphetamine oxidation. Hydrogen peroxide formed at the cytochrome P450 active center plays the key role in hemoprotein inactivation during uncoupling of monooxygenase reactions. The mechanism oxidative heme modification has been studied in monooxygenase reconstituted system also. It was demonstrated that formed at the active center is responsible for the loss of P450 heme and localized outside of active hemoprotein center is responsible for the heme destruction.

The hemoprotein self-inactivation is accompanied by oxidation of all-groups, carbonyl group formation, changes in aggregate state and apoenzyme polymerization. It is probable that heme loss and oxidative modification of apoenzyme is a important step in regulation of P450 decay in cell.

The paper deals with possible mechanisms of cytochrome p-450 self-activation during catalytic turnover. Two routes of hemoprotein inactivation are so far known. The first route studied extensively by many authors, consists in formation of active intermediates capable of modifying heme and apoenzyme. The second route revealed only lately, which results from uncoupled cytochrome P-450-catalyzed monooxygenase reactions, is yet to be clarified. Briefly, it consists in the fact that the hydrogen peroxide formed in the hemoprotein active center interacts with the enzyme-bound Fe2+, thereby generating hydroxyl radicals that bleach the heme and modify the apoenzyme. This mechanism operates with all the substrates and all cytochrome P-450 forms capable of catalyzing the partially coupled monooxygenase reactions proceeding with the formation of hydrogen peroxide as a by-product.

Possible mechanisms of cytochrome P450 self-inactivation during catalytic turnover have been considered. Two ways of hemoprotein inactivation are so far known. The firts, studied extensively by many authors, is the formation of active substrate intermediates, capable of modifying heme and apoenzyme. The second way, revealed quite recently and resulting from uncoupled cytochrome P450-catalyzed monooxygenase reactions, is yet to be clarified. Briefly, it involves formation of hydrogen peroxide in the hemoprotein active center, which interacts with the enzyme associated Fe2+, thereby generating hydroxyl radicals that bleach the heme and modify the apoenzyme. This mechanism operates with substrates and cytochrome P450 forms with partially coupled monooxygenase reactions, thus causing the formation of hydrogen peroxide as a byproduct.

Depending on the localization and functional value, the reactions of the oxidative modification of macromolecules may be classified into two large groups: intracellular and extracellular. To exemplify the first-group reactions, cytochrome P-450 was studied, which is capable of generating different active forms of oxygen during the catalytic cycle. These forms were found to have bactericidal effects and to be able to cause DNA molecule break. In the course of the reaction, cytochrome P-450 also became inactivated under the effect of active oxygen. The involvement of hydrogen peroxide forming directly during peroxycomplex breakdown was proved. It was shown using a soluble reconstructed system that cytochrome P-450 inactivation is attended with the hemoprotein molecule decomposition, destruction and loss of heme, and a rise in the proteolytic vulnerability of the protein. The second reaction group was exemplified by human leucocyte myeloperoxidase which generated hypochlorite along with the active forms of oxygen. Myeloperoxidase showed its bactericidal effect against different strains of bacteria and fungi. The Ames test revealed mutagenic effects of the active oxygen forms generated by myeloperoxidase. In the course of the myeloperoxidase reaction, DNA degradation followed by splitting of the polynucleotide chain and specific pulling out of thymine residues were observed.

Analysis of the effects of inhibitors on NADPH-oxidation in rat liver microsomes shows that the rate of total oxygen consumption parallels the rate of peroxidation of unsaturated fatty acids but not the rate of electron transfer within the chain. The addition of EDTA, 1 mM, to the incubation medium inhibits peroxidation and does not affect NADPH-dependent electron transfer. There are at least three sites for oxygen reduction within the NADPH-oxygenase complex. Oxygen reduction in the chain may occur both at the flavoprotein level and at the level of cytochrome P-450. In addition, Fe 2+ ions bound with the functional groups of electron carriers and catalyzing the peroxidation of unsaturated fatty acids also participate in oxygen reduction. The fact that there are several points of activation of molecular oxygen within the NADPH-dependent electron transport chain precludes the evaluation of the effectiveness of action of inhibitors on cytochrome P-450 by measurement of the total oxygen consumption. NADH oxidation inhibited by EDTA is activated by addition of Ca 2+ .

The absence of correlation between the effect of aniline and aminoantipyrine derivatives on the rates of reduction reactions of cytochrome P-450 and the rate of oxidation of these substrates allows one to suppose that the reductase reaction is not the rate limiting step of hydroxylation for all substrates. Km has been found to be directly proportional to Vmax of hydroxylated substrates. Hence, in these reactions the Km value is not determined by the value Ks but by the (k+2/k+1) ratio and Km does not characterize the affinity of cytochrome P-450 to substrates. Calculations have been made to show that cytochrome P-450 may form two types of enzyme-substrate complexes containing one or two substrate molecules. The complex in which one molecule of cytochrome P-450 binds one substrate molecule is believed to be active.

Data on the stimulating effect of NADH on the rate of NADPH-dependent hydroxylation reactions of dimethylaniline and ethylmorphine favor the possibility of electron transfer from the NADH-oxidation chain to the NADPH-oxidation chain. The presence of the stimulating effect of NADH at a low concentration of NADPH may explain the existence of the low electron-transfer rate from NADH-redox chain to the NADPH-redox chain to the NADPH-redox chain. The exchange of reducing equivalents between the chains occurs behind the flavoprotein sites of the chains.

1. Reactions of N -demethylation, p -hydroxylation and N -oxidation of one substrate, i.e. dimethylaniline, have been used to show that the activating effect of Mg 2+ takes place only in the first two reactions. 2. An increase in V max. of N -demethylation of dimethylaniline is accompanied by an increase in K m . In the p -hydroxylation of dimethylaniline V max. increases whereas K m does not change. A comparison of the changes in the K m values of these reactions with the change in K s shows that in both cases K m does not characterize the affinity of cytochrome P -450 for dimethylaniline. 3. The rate-limiting site of N -demethylation and p -hydroxylation of dimethylaniline, as well as the total rate of NADPH oxidation in the presence of dimethylaniline, is between cytochromes b 5 and P -450. Addition of Mg 2+ to the incubation medium changes the hydrophobic environment of phosphatidylcholine in the membrane, the process being accompanied by a sharp increase in the fluorescence quantum yield of 8-anilinonaphthalene-1-sulphonate.

Definite interaction exists between the induction of microsomal enzymes and induction of the immune response to foreign substances. The loss by phenobarbital of its ability to induce the cytochrome P-450-hydroxylase system when bound covalently with albumin, coupled with the acquisition by phenobarbital of ability to induce a lymphocytic immune system reflects the ability of the two defensive systems of the body — hydroxylase and immune — to interact with foreign compounds of low and high molecular weight. The cytochrome P-450-hydroxylase system of the liver and other tissues is designed to protect the body against the action of low-molecular-weight hydrophobic compounds. The immune system is responsible for the protective effect against high-molecular-weight foreign substances.

A polarographic method is suggested and can be recommended as a simple device for measuring the rates of oxidation of steroid hormones and xenobiotics. The rate of hydroxylation of steroids depends, it is suggested, on the polarity of the molecule of the oxidation substrate.