[Direct detection of an antigen in immunoglobulin G Langmuir-Blodgett films based on a surface plasma resonance method and in a piezoelectric system]. / Priamaia detektsiia antigena v plenkakh Léngmiura-Blodzhett immunoglobulinov G na osnove metoda poverkhnostnogo plazmonnogo rezonansa i v p'ezoélektricheskoi sisteme.

The possibilities of direct antigen detection in unlabelled systems based on immunoglobulin G Langmuir-Blodgett films as a sensitive surface have been studied. It was shown that an increase in the monolayer number in an immunoglobulin G Langmuir-Blodgett film deposited onto a solid surface coated with a thin silver film (50 nm) resulted in the regeneration of the antigen-binding capacity of the upper antibody layer. This dependence can be used for the construction of a direct optical immunosensor based on surface plasmon resonance. Moreover, a model of a piesoelectric immunosensor on the basis of immunoglobulin G Langmuir-Blodgett films for direct ferritin detection has been proposed. The detection range of ferritin concentrations in solution is 10(-10)-10(-7) M.

[IgG, modified with coproporphyrin I and the possibility of direct measurement of the antigen-antibody complex]. / IgG, modifitsirovannyi koproporfirinom I, i vozmozhnost' priamoi registratsii kompleksa antigen-antitelo.

Modification of rabbit IgG by coproporphyrin I activated by Woodward's reagent K as well as antigen-binding and fluorescent properties of coproporphyrin-IgG have been studied. It was shown that coproporphyrin I modified IgG retains its capacity to bind the antigen. The formation of the immune antigen-antibody complex increases the intensity of coproporphyrin-IgG fluorescence.

Functional reconstitution of cytochrome P-450scc with hemin activated with Woodward's reagent K. Formation of a hemeprotein cross-link.

In order to evaluate structure-function relationships of heme moiety in cytochrome P-450scc, we carried out the reconstitution of apoprotein with Fe-protoporphyrin IX, one carboxyl group of which was converted to reactive enol ester by Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate). Woodward's reagent K can be used as a cross-linking reagent, since amino groups can apparently react with the enol ester. Treatment of cytochrome P-450scc with H2O2 was used to obtain the apoprotein. Functional reconstitution of the hemin derivative with apocytochrome P-450scc was achieved. The reconstituted hemeprotein was purified, and the resulting preparation contained no P-420 form and had the same cholesterol-hydroxylating activity as a control preparation. 30% of the reconstituted hemin was covalently bound to protein. Heme-linked peptide (Gly177-Phe194) was isolated. Its possible role in the active site formation of cytochrome P-450scc is discussed.

Immunoassay in the presence of water-soluble polymers.

To overcome the adverse consequences of antibody immobilization (impairment of the antigen-binding parameters) the interaction of a number of antigens with both soluble and covalently immobilized antibodies in the presence of water-soluble polymers has been investigated. Incorporation of some water-soluble polymers (Ficoll, Dextran) into the assay mixture was shown to substantially increase the antigen-binding capacity of both soluble and immobilized antibodies. The use of dextran T70 enhanced the sensitivity of competitive radioimmunoassays for CEA and beta 2-microglobulin 3-4-fold. This procedure may be also applied to shorten the incubation period when performing an assay.

Chemical modification of rabbit IgG with N-dansylaziridine. Investigation of the properties of dansylated antibodies.

To elucidate the effect of antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ), which is sensitive to the changes in the microenvironment, was used for modification of rabbit IgG hinge region cysteine residue. DAZ binds to hinge region Cys 226 as evidenced by the structural analysis. Labelling of IgG with DAZ does not alter its conformation, hydrodynamic behavior, nor its antigen binding properties. Upon antigen bindings, the fluorescence intensity of modified IgG increases about 80%. This result indicates that interaction of antibodies with antigen is accompanied by the conformational changes in the IgG hinge region.

Interaction of cytochrome P-450scc with cytochrome b5.

Spectrophotometric, affinity chromatography and cross-linking experiments provided evidence that cytochrome P-450scc from bovine adrenocortical mitochondria forms a tight complex with cytochrome b5 from rabbit liver microsomes. In the reconstituted system cholesterol side chain activity of cytochrome P-450scc was enhanced by the addition of cytochrome b5.

[Chemical modification of rabbit immunoglobulin G by dansylaziridine and study of the properties of modified antibodies]. / Khimicheskaia modifikatsiia immunoglobulinov G krolika N-dansilaziridinom i issledovanie svoistv modifitsirovannykh antitel.

To elucidate the effect of the antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ) which is sensitive to microenvironmental changes, was used for modification of the rabbit IgG hinge region cystine residue. DAZ binds to the hinge region Cys 226 as could be evidenced from the structural analysis data. Labelling of IgG with DAZ does not alter either its conformation and hydrodynamic behaviour or its antigen binding properties. Upon antigen binding the fluorescence intensity of modified IgG increases up to about 80%. This finding suggests that the interaction of antibodies with the antigen is accompanied by conformational changes in the IgG hinge region.

Is cytochrome P-450scc a transmembrane protein?

The topology of cytochrome P-450scc in the inner mitochondrial membrane of adrenal cortex has been investigated using monospecific antibodies to cytochrome P-450scc and its fragments F1 (Ile1-Arg250), F2 (Asn257-Ala481) and F3 (Asn257-Arg399). Antibodies to F1 and F2 were shown to effectively bind to the matrix and cytosolic sides of the inner membrane. Antibodies to F3 specifically interacted only with the matrix side of the membrane. These data are consistent with a model of molecular organization which shows that cytochrome P-450scc is a transmembrane protein, both N- and C-terminal sequences of the cytochrome being able to span the membrane.

[Reconstitution of the cholesterol-hydroxylating system from adrenal cortex mitochondria by cytochrome P-450scc, adrenodoxin and the redox-mediator neutral red]. / Rekonstruktsiia kholesteringidroksiliruiushchei sistemy iz mitokhondrii kory nadpochechnikov na osnove tsitokhroma P-450scc, adrenodoxina i redoks-mediatora neitral'nogo krasnogo.

It has been found that 3-amino-7-dimethylamino-2-methylphenazine (neutral red, NR) is responsible for the electron transport from the cathode to adrenodoxin (Ad) and cytochrome P-450 (P-450scc) from adrenal cortex mitochondria inaccessible to direct electrochemical reduction under native conditions. The rate constant for Ad reduction by this mediator is equal to 1.1 x 10(5) M-1 s-1 at 25 degrees C; the values of enthalpy and entropy for the activation reaction are 26.6 kJ.mol-1 and -59.6 J.mol-1 deg-1, respectively. Using the shunted electron transport chain NR----Ad----P-450scc, the cholesterol conversion into pregnenolone in an electrochemical cell was performed. Pregnenolone was found to be the sole steroid product of this reaction. Superoxide dismutase and catalase had no effect on the activity of the shunted system. After removal or substitution of Ad the apo-Ad hemoprotein was reduced in a non-productive manner. Under identical reconstitution conditions methylviologen was ineffective as an electron carrier.

[The radioimmunological determination of human placental lactogen with the RIO-PL-125I reagent kit]. / Radioimmunologicheskoe opredelenie platsentarnogo laktogena cheloveka naborom reaktivov RIO-PL-125I.

The authors described the development of a radioimmunoassay for the determination of human placental lactogen (PL) in the blood serum of pregnant women. Methods of obtaining RIA components were developed: highly refined and stable agents of the labeled and unlabeled hormones, specific antisera and a highly effective separating agent. The test system and commercial kits were used for comparative determination of the blood PL concentration at various terms of pregnancy. Similar results were obtained. The above test system was laid in the basis of a commercial kit RIO-PL-125I (USSR).

[Chemical modification of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria with diethylpyrocarbonate]. / Khimicheskaia modifikatsiia kholesteringidroksiliruiushchego tsitokhroma P-450 mitokhondrii kory nadpochechnikov diétilpirokarbonatom.

Chemical modification of adrenocortical cytochrome P-450scc with diethyl pyrocarbonate has been carried out. The histidine residues and the protein amino groups were shown to undergo modification. Carbethoxylation was accompanied by the hemoprotein inactivation and the loss of enzymatic activity. Neither of the high spin effectors (i.e., substrate and adrenodoxin) protected cytochrome P-450scc either from inactivation or from the loss of enzymatic activity. The data obtained are discussed in terms of the functional role of histidine residues in the cytochrome P-450scc molecule.

Immunochemical analysis of adrenocortical cytochrome P450scc: 1. Monoclonal antibodies against haemprotein.

Twelve stable mouse hybridoma cell lines producing monoclonal antibodies against bovine adrenocortical cytochrome P450scc were prepared. All the monoclonal antibodies interacted specifically with the antigen as shown by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), dot-immunoblotting, and Western blot analysis, but did not precipitate in Ouchterlony double-immunodiffusion analysis. A highly sensitive competitive RIA based on monoclonal and polyclonal mouse antibodies was developed to determine the total content of P450scc in adrenocortical mitochondria. Three of the monoclonal antibodies strongly inhibited the conversion of cholesterol to pregnenolone.

[Chemical modification of antibodies against carcino-embryonal antigen]. / Khimicheskaia modifikatsiia antitel protiv rakovo-émbrional'nogo antigena.

Chemical modification of different amino acid residues and the carbohydrate moiety of antibodies to carcino-embryonic antigen (CEA) was used to elucidate their role in the interaction with CEA and to evaluate the effect of antibody modification and steric factor in immunosorbent synthesis. The distribution of the modified groups among the structural fragments of IgG and the levels of changes in the antigen-binding properties of modified antibodies were investigated. The Fc-fragment of IgG was shown to contain carboxylic groups, tyrosine and histidine residues, whose modification influences the antigen-binding properties as a result of generalized conformational changes in the IgG molecule. A comparison of these results with earlier obtained data on the functional properties, of immobilized antibodies revealed that the decrease of antigen-binding characteristics of anti-CEA after IgG immobilization via NH2- and COOH-groups, carbohydrate moiety, tyrosine and histidine residues is due to the direct effect of antibody modification, whereas the changes in parameters of antibody interaction with antigen after IgG immobilization via SH-groups, methionine and histidine residues is due to steric hindrances.

Inhibitory domain-specific antibodies to cytochrome P-450scc.

Highly specific antibodies to cytochrome P-450scc and its F1 and F2 fragments, representing N- and C-terminal sequences of the hemeprotein respectively, were raised in rabbits. These antibodies were found to be inhibitory (up to 50-90%) for the cholesterol transformation into pregnenolone in the reconstituted system, indicating the involvement of both F1 and F2 domains formed by the respective fragments in monooxygenase catalysis. Cytochrome P-450scc in mitoplasts is not accessible for trypsin as revealed by immunological techniques. However, the treatment of submitochondrial particles with trypsin results in two main fragments identified by immunoblotting in the presence of the monospecific antibodies as F1 and F2 fragments. This indicates that the trypsin sensitive 250-257 region in cytochrome P-450scc molecule connecting both domains is exposed to the matrix side of the inner mitochondrial membrane.

Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin.

Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.

[Study of the role of lysine residues of the cholesterol hydroxylating cytochrome P-450 by a method of chemical modification]. / Issledovanie roli ostatkov lizina kholesteringidroksiliruiushchego tsitrokhroma P-450 metodom khimicheskoi modifikatsii.

Chemical modification of cytochrome P-450scc by lysine-specific reagents has been performed. Modification of the hemoprotein was shown to result in the loss of its ability to interact with adrenodoxin. With a view of identifying lysine residues involved in the interaction with adrenodoxin, cytochrome P-450scc was modified by succinic anhydride in the presence of adrenodoxin. After the removal of ferredoxin, the modification was performed with the use of a radioactively labeled reagent. Subsequent hydrolysis of the succinic hemoprotein by chymotrypsin and separation of the peptides obtained by high pressure liquid chromatography resulted in the isolation of seven chymotryptic peptides containing labeled lysine residues. These amino acid sequences were identified. The role of lysine residues of cytochrome P-450scc in complex formation with adrenodoxin is discussed.

[Immunochemical study of cholesterol-hydroxylating cytochrome P-450. Topology of the peptide chain of the heme protein in the phospholipid membrane]. / Immunokhimicheskoe issledovanie kholesteringidroksiliruiushchego tsitokhroma P-450. Topologiia polipeptidnoi tsepi gemoproteida v fosfolipidnoi membrane.

The topology of adrenocortical cytochrome P-450scc in inner mitochondrial membrane was studied. To determine the polypeptide chain parts exposed to matrix or cytosol, two approaches were used, i.e. i) limited proteolysis of membrane-bound cytochrome P-450scc followed by the detection of the peptides formed by immunoblotting; ii) binding of monospecific antibodies against cytochrome P-450scc as well as fragments F1 and F2 representing N- and C-terminal sequences of the hemeprotein, to membrane structures (mitoplasts and submitochondrial particles). The data obtained confirm the transmembrane orientation of cytochrome P-450scc molecule, since antibodies against the hemeprotein as well as fragments F1 and F2 were found to be bound both on the matrix and cytosol surfaces of the inner mitochondrial membrane. It was shown that region 250-257 in cytochrome P-450scc connecting domains F1 and F2 is exposed to the matrix. A model of molecular organization of cytochrome P-450scc in inner mitochondrial membranes is proposed.

Cross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria.

Cytochrome P-450SCC and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of cross-linked complex and 6% of free cytochrome P-450SCC was obtained after purification on cholate-Sepharose. Cytochrome P-450SCC in cross-linked complex completely preserves its high-spin form in the presence of Tween 20 or pregnenolone. Utilization of radioactively labelled adrenodoxin, chemical cleavage of cytochrome P-450SCC from cross-linked complex with o-iodosobenzoic acid and HPLC for separation of peptides allow us to conclude that the complex of cytochrome P-450SCC with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450SCC-Leu-88-Thr-107 and Leu-368-Gly-416. The cross-linked complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450SCC with an apparent molecular mass of 114 kDa was obtained with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. The composition of cross-linked complex was determined by immunoblotting and by evaluation of radioactivity using preliminary N-ethyl[2,3-14C]maleimide-modified adrenodoxin. From this data it appears that the ternary complex may exist in solution.

[Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5]. / Vzaimodeistvie kholesteringidroksiliruiushchego tsitokhroma P-450 s tsitokhromom b5.

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.

[A radioimmunologic test system for determining beta 2-microglobulin in human blood serum and urine]. / Radioimmunologicheskaia test-sistema dlia opredeleniia beta 2-mikroglobulina v syvorotke krovi i moche cheloveka.

The authors described a radioimmunoassay developed for the determination of beta 2-microglobulin (beta 2-M) concentration in human serum and urine using 125I-labeled beta 2-M. This method permitted the determination of beta 2-M concentration within 0.5-50 mg/l in the blood serum and within 0.02-50 mg/l in urine. beta 2-M concentration determined by this assay, was 1.71 +/- 0.58 mg/l in the blood serum and 0.097 +/- 0.32 mg/l in the urine of healthy persons.