[Atomic force microscopy monitoring of temperature dependence of cytochrome BM3 oligomeric state].

The change in temperature is one of the factors affecting the activity of enzymes. In this work thermal denaturation and aggregation of cytochrome P450 BM3 were studied by atomic force microscopy. To determine specific temperature transitions the fluorescence analysis was used. In the low melting temperature range, 10-33 degrees C, a decrease in the fluorescence intensity of aromatic residues was observed with an increase in the fluorescence intensity of flavin groups. Protein melting in this range indicated three narrow S-shaped cooperative transitions at temperatures 16, 22 and 29 degrees C. Atomic force microscopy analysis in this temperature range showed that the shape of BM3 molecules remained globular in the form of compact objects (heights h < 7 nm, lateral dimensions d < 50 nm), but protein oligomeric state changed. The first two transitions were accompanied by a decrease in the degree of oligomerization and the third one was accompanied by its increase.

[Irreversible chemical AFM-fishing for the detection of low-copied proteins].

The atomic-force microscopy-based method of irreversible chemical AFM-fishing (AFM-IF(Ch)) has been developed for the detection of proteins at ultra-low concentrations in solution. Using this method, a very low concentration of horseradish peroxidase (HRP) protein (10(-17) M) was detected in solution. A theoretical model that allows the description of obtained experimental data, is proposed. This model takes into consideration both the transport of the protein from the bulk solution onto the AFM-chip surface and its irreversible binding to the activated area.

[Oligomeric state investigation of flavocytochrome CYP102A1 using AFM with standard and supersharp probes].

Atomic force microscopy with two types of probes - standard (radius of curvature R approximately 10 nm) and supersharp (R approximately 2 nm)- was used to determine CYP102A1oligomeric state. CYP102A1 images were obtained in a liquid, air and vacuum environment using the standard probes, also a ratio of monomers to oligomers (alpha) of CYP102A1 were determined as alpha=0.48:0.52. At the same time use of standard probes did not allow to resolve the structure of these oligomers. Supersharp probes allowed to obtain the data about the monomers to oligomers ratio, and also about the dimers/trimers/tetramers ratio in air and vacuum. So, a ratio alpha of CYP102A1 in liquid can be determined by the standard probes, and an oligomeric state of protein can be specified by the supersharp probes.

[Atomic force microscopy visualization and measurement of the activity and physicochemical properties of single monomeric and oligomeric enzymes].

An approach to measure the activity of single oligomers of the heme-containing enzyme the cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22 degrees C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule reduced to the unit time was 5+/-2 Ac. The elasticity of a single protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made i t possible t o determine t he integral andlocal Young's modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37+/-117 and 1+/-3 MPa, respectively.

[Mass-spectrometric identification of interaction sites between cytochrome P450 2B4 and NADPH cytochrome P450 reductase].

We determined the interaction sites of the cytochrome P450's protein-partners: 2B4 (d-2B4) and NADPH-cytochrome P450 of reductase (d-Fp). While in operation, these proteins are forming the complexes. We used 4-4'-dithio(bisphenyl)azide linker for non-specific covalent coupling of d-2B4 complexes with d-Fp in Emulgen-913-monomerized system. Covalently-linked peptides in this complex were identified with ESI-MS/MS. Several sites of these proteins' binding with each other were revealed. Based on them, a model of intermolecular protein interactions was created. The model includes 5 cross-linker-stabilized contact sites of d-2B4 with d-Fp involving the following peptides of d-2B4 and d-Fp: (1) d-2B4423-433 and d-Fp 102-109; (2) d-2B4324-336 and d-Fp570-585; (3) d-2B4327-336 and d-Fp452-464; (4) d-2B4 192-197 and d-Fp456-464; (5) d-2B4 134-139 and d-Fp406-425. Herein, in the latter two cases, the peptides of d-Fp are located in their inter-domain slit and stabilize protein-protein complex via nanoprobe cross-linker; therefore, the formation of d-2B4/d-Fp complexes in these sites may involve aminoacid residues d-Fp456-464 and d-Fp406-425 surrounding inter-domain slit.

[Visualization and identification of hepatitis C viral particles by atomic force microscopy combined with MS/MS analysis].

Possibility of detection and identification of hepatitis C viral particles with mass spectrometry (MS) in combination with atomic force microscopy (AFM) had been investigated. AFM/MS approach is based on two technologies: (1) AFM-biospecific fishing that allows to detect, concentrate from solution and to count protein complexes on a surface of AFM-nanochip; (2) mass spectrometric identification of these complexes. AFM-biospecific fishing of HCVcoreAg from solution was carried onto surface of AFM-nanochips with immobilized anti-HCVcoreAg. It was shown that HCVcoreAg/anti-HCVcore(im) complexes were formed onto AFM-nanochips in quantity sufficient for mass spectrometric identification. Thus, AFM/MS approach allows to identify fragments of hepatitis C virus fished onto a surface of AFM-nanochip from serum.

[Productive and non-productive complexes in cytochrome P450-containing system].

The equilibrium dissociation constants K(D), the complex association / dissociation rate constants (k(on)/ k(off)) and the lifetimes of redox partners' complexes were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450 2B4 and P450scc). To estimate the productivity of complexes formed within the systems studied, the Q parameter--i.e. the ratio of protein-protein complex lifetime (T(LT)) to the time required for a single hydroxylation cycle (tau(cat))--was determined. It was shown that Q was changed (albeit insignificantly) upon transition from the oxidation to hydroxylation conditions in all the three P450 monooxygenase systems studied. It was shown that the binary complexes formed within the P450cam and the P450scc systems requiring an intermediate electron-transfer protein between the reductase and cytochrome P450 were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such an intermediate electron-transfer protein, proved to be productive. Formation of ternary complexes within the three systems was demonstrated under hydroxylation conditions. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were virtually 100% productive. Within the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.

[Atomic force microscopy detection of serological markers of viral hepatites B and C].

The aim of the study was to demonstrate the possibility of detection of serological markers, containing the hepatitis B surface antigen (HBsAg) and hepatitis C virus core-antigen (HCVcoreAg) in human serum, by use of a new atomic force microscopy (AFM)-based nanotechnological approach. In this study, the immobilization on AFM-chip of antibodies against the hepatitis B virus surface antigen (anti-HBsAg) as well as the antibodies against the hepatitis C virus core antigen (anti-HCVcoreAg) was performed. It was shown that such approach enables to detect: HBsAg, HCVcoreAg and the viral fragments containing these antigens in the serum. Comparative analysis of detection of HBsAg- and HCVcoreAg-containing particles by use of the AFM method vs. traditional methods (ELISA, PCR) has demonstrated the 75% coincidence of results between the AFM and the latter two methods.

[The optical biosensor study of the redox partners interactions within the cytochrome P450 2B4-containing monooxygenase system in hydroxylation conditions].

Interactions between cytochrome P450 2B4, NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH, in the monomeric reconstituted P450 2B4-contained monooxygenase system. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of an optical biosensor IAsys+. It was shown that, despite immobilization of any of the partners (via their respective amino groups) onto the carboxymethyldextran surface of the IAsys+ optical biosensor, its activity didn't loss. The formation of binary d-Fp/d-2B4 complexes was registered. The association/dissociation rate constants (kon/koff) were (0,013 +/- 0,005) x 10(6) M(-1) x s(-1)/0,05 +/- 0,02 s(-1), equilibrium dissociation constant (K(D)) was (0,26 +/- 0,13) x 10(-6) M. Comparison of kon, koff and K(D) for d-Fp/d-2B4 complexes in oxidation conditions with corresponding constants for the oxidized protein forms--(0,10 +/- 0,03) x 10(6) M(-1) x s(-1)/(0,14 +/- 0,06) s(-1), (0,71 +/- 0,37) x 10(-6) M--shows that the decrease in kon and K(D) occurs due to the increase in lifetime during transition from oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in oxidation and hydroxylation conditions. The ternary d-Fp/d-2B4/d-b5 complexes formation was shown in hydroxylation and oxidation conditions.

[Interaction of pyruvate kinase with isatin and deprenyl].

The glycolytic enzyme, pyruvate kinase, exhibits moderate affinity [3H]isatin binding (KD approximately 10 microM), which is inhibited by ATP (IC50 25 microM) and deprenyl (IC50 5 microM). Interaction of pyruvate kinase with isatin and its inhibition by ATP and deprenyl has also been confirmed using an independent biosensor technique and immobilized isatin analogue, aminoisatin. This effect has some specificity because the enzyme, creatine phosphokinase, does not exhibit specific isatin-binding. It is suggested that interaction of pyruvate kinase with isatin may reflect some non-glycolytic functions of this enzyme.

[Nanobiotechnology and nanomedicine].

Nanobiotechnology is a new direction in the technological science, which plays a key role in creation of nanodevices for analysis of living systems on a molecular level. Nanomedicine is the application of nanotechnologies in medicine for maintenance and improvement of human life using the knowledge on human organism at a molecular level. Application of nanoparticles and nanomaterials for the diagnostic and therapeutic purposes is now significantly extended in nanomedicine. Use of nanotechnological approaches and nanomaterials opens new prospects for creation of drugs and systems for their directed transport. Implementation of optico-biosensoric, atomic-force, nanowire and nanoporous approaches into genomics and proteomics will significantly enhance the sensitivity and accuracy of diagnostics and will shorten the time of diagnostic procedures that will undoubtedly improve the efficiency of medical treatment. The review highlights data on application of nanobiotechnologies in the field of diagnostics and creation of new drugs.

[Optical biosensor study and molecular modeling of interactions between cytochrome P450cam and cytochrome b5].

The formation of complexes of cytochrome P450cam (P450cam) with full-length cytochrome b5 (d-b5) and its tryptic water-soluble fragment (t-b5) was analyzed using a two-channel IAsys+ optical biosensor. It was found that t-b5 can form complexes with P450cam, while d-b5 does not interact with P450cam. The involvement of amine groups of P450cam in the complex formation was demonstrated. The temperature dependence of t-b5(im)/P450cam complex formation was measured. The association rate constant (k(on)) increased with temperature, while the dissociation rate constant (k(off)) practically remained unchanged. It was concluded that hydrophobic interactions play a key role in the complex formation, while electrostatic interactions are significant for complex stabilization. Based on temperature dependence the activation energy, enthalpy and entropy of complex formation were calculated. It was shown that the entropy component plays a key role in t-b5(im)/P450cam interaction. Computer modeling of P450cam/t-b5 and P450cam/d-b5 interactions was carried out. Using the method of molecular docking some hypotheses of protein-protein complexes were advanced and the best ones were selected based on geometric complementarity, calculated binding energy and probability of electron tunneling between proteins. The computer modeling has shown that only P450cam and t-b5 can form the stable complex. These results are in good agreement with the experimental data obtained with the optical biosensor.

[Optical biosensor study of interaction between trypsin and trypsin inhibitor].

The formation of complexes between trypsin (T) and soybean tripsin inhibitor (STI) was analyzed, using a two-channel IAsys+ optical biosensor. The temperature dependence of complex formation (with its major parameters--the association rate constants (k(on)), the dissociation rate constants (k(off)) and the equilibrium constants (Kp)) was determined. The equilibrium constants obtained well correlate with those obtained by other methods. The association rate constant for the binding of T/STI complex increased with temperature, while the dissociation rate constant practically remained unchanged. The association and dissociation rate constants activation parameters were calculated from their concentration dependence. Based on the temperature dependence the activation energy, enthalpy and entropy of complex formation were calculated. It was shown that the entropy component plays a key role in T/STI interaction.

[Detection of hepatitis B virus surface antigen with the use of an optical biosensor]. / Opredelenie poverkhnostnogo antigena virusa gepatita B s pomoshch'iu opticheskogo biosensora.

The detection of hepatitis B virus surface antigen (HBsAg) with the use of a model IAsys+ two-channel optical biosensor is based on the registration of interaction between anti-HBs monoclonal antibodies forming the surface layer of the biochip of the biosensor cuvette and blood serum HBsAg. For the first time a two-channel optical biosensor has been used for the detection of HBsAg in blood serum samples. The comparative analysis of the detection of HBsAg by two methods, viz. with the use of an optical biosensor and the enzyme immunoassay, has demonstrated lower sensitivity, but higher specificity of the detection of this antigen by means of a model IAsys+ biosensor with the biochip, prepared in the process of the work. The main advantages of the biosensor detection lie in the registration of interaction in real time without introducing special markers into the molecules under study.

[Glycerol-3-phosphate dehydrogenase--cytosolic isatin-binding protein]. / Glitserol-3-fosfatdegidrogenaza--izatin-sviazyvaiushchii belok tsitozolia.

Isatin is an endogenous indole widely distributed in mammalian tissues and body fluids. The presence of isatin-binding proteins has been recognised in particulate and soluble fractions of various organs and tissues. However, identified targets of isatin action (monoamine oxidase, natriuretic peptide receptor type A and soluble NO-stimulated guanylate cyclase) cannot account for all biological activity of this compound. Highly purified glycerol-3-phosphate dehydrogenase (GPDH) from rabbit muscle effectively interacts with the isatin analogue immobilised on the cuvette of IAsys optical biosensor. This effect was specific because the other NAD-dependent cytosolic enzyme purified from rabbit muscle, lactate dehydrogenase failed to interact with the immobilised isatin analogue. Replacement of the cuvette medium for washing buffer did not cause total dissociation of GPDH-isatin complexes. This suggests involvement of several types of enzyme-isatin interaction including tight binding. Low (10 microM) and high (100 microM) concentrations of isatin caused different effects on GPDH activity: the former significantly increased apparent Km for NAD, whereas the latter decreased apparent Vmax and increased Km.

[Study of the tissue and subcellular distribution of isatin-binding proteins with optical biosensor]. / Issledovanie tkanevogo i subkletochnogo raspredeleniia izatin-sviazyvaiushchikh belkov pri pomoshchi opticheskogo biosensora.

An original method for the integral evaluation of tissue and subcellular distribution of isatin binding proteins has been developed. This method is based on continuous monitoring of changes of optical characteristics that accompany complex formation between a ligand (immobilized on dextran bed of IAsys biosensor cell) and its soluble receptor. Solubilisation of tissue preparations and subcellular fractions with detergent (1% Triton X-100) is the important preconditions for the applicability of this method. The immobilisation of 5-aminoisatin was achieved by peptide bond formation between amino group of this isatin analogue and carboxyl group of the dextran bed of the biosensor cell. Addition of Triton X-100 treated preparations of membrane and soluble fractions of rat brain, liver, heart, and kidneys to the biosensor cell resulted in appearance of the characteristic response, indicating complex formation with the immobilised isatin analogue. The magnitude and a shape of kinetic curve vary in these samples. Isatin binding proteins predominated in membrane fractions of brain, liver and heart preparations whereas in the kidneys the highest isatin-binding response was detected in the soluble fraction. The distribution of isatin binding sites in the particulate fraction reduced in the following order: brainstem > brain hemispheres = cerebellum > heart > kidneys > liver. In the soluble fraction there was different rank of isatin binding activity: kidneys > heart > brainstem = brain hemispheres > liver > cerebellum. Liver outer mitochondrial membranes are characterised by the higher isatin-binding than mitochondria. Treatment of mitochondria with clorgyline and deprenyl, specifically inhibiting MAO A and B, respectively, significantly reduced the magnitude of the biosensor response and changed the shape of the kinetic curve. These data are consistent with the notion that within mitochondria MAOs are the major targets of isatin.

[Detection of the hepatitis B virus surface antigen with a biosensor]. / Detektsiia poverkhnostnogo antigena virusa gepatita B s pomoshch'iu opticheskogo biosensora.

A new method for detection of hepatitis B surface antigen (HBsAg) has been developed. It employees IAsys optical biosensor registration kinetics of HBsAg complexes with monoclonal antibodies. Detection of intermolecular interactions is accompanied by changes of the light refraction coefficient in the sensitive layer of the biosensor cuvette. The main advantage of this diagnostic technique consists in rapid registration of these interactions in real time, without any introduction of special labels into analysing molecules. The optical biosensor method was successfully employed for the detection of HBsAg in human blood serum. A comparative study of HBsAg detection by the optical biosensor and by immunoenzyme analysis demonstrated high specificity of HBsAg detection by this new method.