[Functionalized Metal Chelates Based on Diethylenetriaminetetraacetic Acids for Chemical Modification of Proteins and Small Biomolecules].

Bifunctional reagents based on diethylenetriaminetetraacetic acid containing a bound metal ion and a reactive functional group for the interaction with proteins and low-molecular-weight substances have been synthesized. An Amino-derivative of a complexonate was obtained by acylation of monosubstituted diamine with diethylenetriaminepentaacetic acid dianhydride followed by deprotection ofthe amino group, purification by anion exchange chromatography and chelation of Eu3+. This metal chelate derivative was used for labeling 17α-hydroxyprogesterone 3-(O-carboxymethyl)oxime and horseradish peroxidase. The enzyme modified with the Eu3+ complexonate at the carbohydrate component and with a cortisol derivative at the polypeptide chain was used in a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) as well as in an enzyme immunoassay of the steroid hormone. DELFIA showed that labeled 17α-hydroxyprogesterone retained the affinity for corresponding antibodies. A Eu(3+)-complexonate carboxy-derivative N-succinimide ester was obtained by acylation of the aminochelate with p-phthalic acid di-N-succinimide ester. It was used for modification of amino groups of lysine residues in polypeptide chains of human serum albumin and some immunoglobulins G. Purification of Eu3+ complexonate-protein conjugates by gel-chromatography on a Superose- 12 column allowed to separate the modified proteins from unreacted low molecular weight Eu(3+)-derivatives and to determine a degree of lanthanide inclusion into a protein. The amount of Eu3+ covalently attached to a protein was determined by measuring the fluorescence of a conjugate in the dissociative-enhancement solution. The obtained values correlated well with the results of ICP-MS determination of Eu3+ concentration in a conjugate solution. It was shown that conjugates of monoclonal antibodies obtained by the proposed method possessed the required characteristics of fluorescence intensity, signal-to-noise ratio, sensitivity and specificity in DELFIA medical diagnostic systems.

[Interaction of triiodothyronine-biotin conjugate with binding proteins in immunoassay systems].

A new construction of a test-system for free thyroid hormone 3,3',5-triiodothyronine determination (free T3) in human blood serum in ELISA and IRMA variants was described. For this purpose a low-molecular weight bifunctional conjugate containing T3 and vitamin H (biotin, Bt) residues was synthesized. The conjugate (T3-Bt) can be bound via its biotin function to biotin-binding proteins on a solid phase whereas its T3-portion can interact with monoclonal antibody against T3 (anti-T3-MAb) labeled with horseradish peroxidase or iodine-125 in an enzyme immunoassay or an immunoradiometric assay system (ELISA or IRMA), respectively. The immunochemical interaction is hindered if the T3 residue takes part as a component of a preformed solid phase complex between T3-Bt and avidin. Computer modeling shows that in this complex glycan component of avidin shields iodothyronine residue. The binding of T3-Bt both with T3-free sites on anti-T3-MAb and the solid phase avidin achieves with help of a delayed T3-Bt addition into avidin-covered containers with preliminary co-incubated labeled anti-T3-MAb and an assayed T3 sample.

The role of components of Bifidobacterium and Lactobacillus in pathogenesis and serologic diagnosis of autoimmune thyroid diseases.

During recent years, researchers have been focusing on the concept of an infectious etiology of autoimmune diseases. The most discussed theory is molecular mimicry, i.e. the emergence of autoreactive clones of T- and B-lymphocytes as a result of cross-immune response to homologous bacterial or viral antigen. Information on the role of probiotic microorganisms (PM) in the molecular mechanisms of autoimmune thyroid diseases (ATD) is limited. Using proteins and immunogenic peptides databanks and relevant computer programs, the homology between the amino acid sequences of thyroid peroxidase (TPO) and thyroglobulin (Tg), which are potential B- and T-cell epitopes of these antigens, and proteins of bifidobacteria and lactobacilli was established. Moreover, we have found components of cells of Bifidobacterium bifidum 791, Bifidobacterium adolescentis 94 BIM, Bifidobacterium longum B379M and Lactobacillus plantarum B-01 that selectively bind human antibodies to TPO (anti-TPO) and antibodies to Tg (anti-Tg) and compete with natural antigens for the binding of anti-TPO and anti-Tg in ELISA. Additionally, a three-fold difference was observed between the probability of detecting antibodies (Abs) to the antigens of L. plantarum B-01 and B. bifidum 791 in serum samples containing and those not containing anti-TPO. On the whole, our data are arguments in favour of the assumption of the possible role of PM of the genera Bifidobacterium and Lactobacillus in triggering ATD by the mechanism of molecular mimicry. The data obtained in silico and in vitro should be proven by use of animal models and clinical studies for extrapolations to the whole body. Possible antigenic properties of components/proteins of bifidobacteria and lactobacilli, selectively binding anti-TPO and anti-Tg should be taken into consideration. Natural human Abs to these bacterial components are probably able to cross-react with the TPO and Tg in the ELISA for detection of anti-TPO and anti-Tg, which are serologic markers of ATD. It can lead to unspecific false positive results and, hence, to an incorrect diagnosis.

[The biotin-thyroxin conjugate as a bifunctional ligand of binding proteins].

The conjugate of the residue of vitamin H (biotin, Bt) with the hormone of thyroid gland thyroxin (T4) was prepared by N-acylation of N-(3-aminopropyl) biotin amide with N-hydroxysuccinimide ester of N-acetyl thyroxin. The interactions of the Bt-T4 conjugate with one or simultaneously with two binding proteins with affinity to Bt or T4 in solution and on a solid phase were studied by electron spectroscopy, enzyme immunoassay, and computer modeling. Bt-T4 was specifically fixed in the Bt-binding site of the streptavidin molecule via a large number of hydrogen bonds and hydrophobic interactions. The maximum of the streptavidin fluorescence shifted to a long-wave area and its intensity decreased as a result of complex formation. The degree of quenching of the protein emission was significantly higher than that of the streptavidin-Bt complex. Additional fluorescence quenching resulted from interactions which were sensitive to pH, ionic strength, and detergents and stabilized the position of the thyroxin part of the conjugate near Trp120 of streptavidin in its complex with Bt-T4. The Bt-T4 conjugate also formed a specific equimolar complex with T4-binding human globulin (TBG) by the same mechanism as that for T4. The Bt residue did not participate in the interactions which changed characteristics of the TBG fluorophores. The Bt-T4 conjugate was bound to avidin on a solid phase in the solid phase enzyme immunoassay owing to its biotin function, whereas its thyroxin part was exposed to a solution and interacted with polyclonal antibodies to T4. The intact T4 competitively inhibited this interaction after its addition to the system. Bt-T4 also exhibited its bifunctional activity in other immune analytic system. The conjugate bound streptavidin was labeled with Eu(3+)-chelate and subsequently formed a three component complex with participation of a monoclonal antibody to T4 immobilized on a solid phase. Free T4 inhibited the thyroxin function of the conjugate bound to the labeled streptavidin proportionally to its concentration in a sample of human blood serum. Parameters of the immunofluorescent analysis demonstrated that the streptavidin-Bt-T4 complex was actively bound to the T4-antibody, but had practically no interaction with serum T4-binding proteins, including TBG. Probably, nonspecific interactions of the T4 residue with streptavidin in its complex with Bt-T4, along with steric factors, complicated penetration of thyroxin in this complex into active sites of TBG and other T4-binding proteins of blood serum. The Bt-T4 stable conjugate was synthesized according to a plain scheme and could be used as a bifunctional ligand of binding proteins in biochemical studies and immune analytical systems for medicinal diagnostics.

[Precipitation of streptavidin complexes with biotinylated proteins in agar gel].

The effect of precipitation of complexes of streptavidin with biotinylated proteins under conditions of simple (according to Mancini) and double (according to Ouchterlony) radial diffusion in agar gel was studied. The position and form of precipitation lines depended primarily on the initial concentration of components and the degree of protein biotinylation. Free biotin, 1% SDS, and 6 M urea contained in the gel, as well as thermal denaturation of streptavidin inhibited the precipitate formation. Mannose, glucose, fucose, galactose, sucrose, and NaCl at high concentrations had no effect on biospecific precipitation. A model of interaction of streptavidin with biotinylated macromolecules is suggested, which accounts for the observed effect, and the prospects of practical application of the precipitation effect are discussed.

[Enzyme immunoassay of (24R)-brassinosteroids].

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.

[Luminescent analysis of the structure of human alpha-1-microglobulin].

The spectra of absorption, fluorescence, and excitation of fluorescence of preparations of alpha-1-microglobulin isolated from human urea by two methods, gel chromatography and immunoaffinity chromatography with additional purification by activated charcoal, have been investigated in ultraviolet and visible regions. A possible nature of low-molecular compounds coloring alpha-1-microglobulin yellow-brown and their role in stabilizing the structure of protein globule are discussed. The action of urea (1.0-10 M) and guanidine hydrochloride (0.25-6 M) on the conformational state and the fast (nanosecond) internal dynamics of alpha-1-microglobulin has been investigated by the method of tryptophan fluorescence. It has been shown that the unfolding of alpha-1-microglobulin under the action of these denaturants is associated with a significant increase in the nanosecond internal dynamics of protein. The ability of alpha-1-microglobulin to restore the initial conformation characteristic for the native protein and the internal dynamics after the unfolding of the globule by 10 M urea and 6 M guanidine hydrochloride has been ascertained. It has been found that alpha-1-microglobulin isolated by the method of gel chromatography can exist in solution of 4-6 M urea in a thermodynamically stabile partialy folded state.

[Characteristics of immunoaffinity chromatography and a new method for isolation of human thyroid peroxidase from subcellular fractions of thyroid gland].

A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. Immunochemical properties of TPO were studied under variable conditions, and a new method for isolating the protein from microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immolbilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.

[Characteristics of monoclonal antibodies against human thyroid peroxidase for use in immunoaffinity chromatography and immunoassays].

Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (Ka values are in the range 10(8)-10(9) M(-1)). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).

[Stabilization of diluted aqueous solutions of horseradish peroxidase]. / Stabilizatsiia razbavlennykh vodnykh rastvorov peroksidazy khrena.

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55 degrees C and infinite dilution, HRP was inactivated with a rate constant of 2.86 x 10(-3) s-1. CaCl2, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the "glycerol-based" one containing 10% ethanol and 20% glycerol, or the "protein-based" one containing 0.1% Kathon CG and 0.2 g/l of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl2 were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.

[Autoantibodies to human thyroid peroxidase in immunoassay and immunoaffinity chromatography]. / Autoantitela k tiroperoksidaze cheloveka v immunoanalize i immunoaffinnoi khromatografii.

A biochemical system of radioimmunoassay of human thyroid peroxidase (TPO) with the use of specific autoantibodies was developed for the first time. This system includes lyophilized preparations of human autoantibodies to TPO, radioiodinated TPO, standards prepared from pure TPO, and the solid-phase protein A as a precipitating agent. An analytical system was used to study the TPO distribution in fractions of thyroid tissue homogenate. Differences in TPO concentrations in mitochondria, microsomes, and supernatant fluid depending on thyroid pathology and tissue storage conditions were found. The behavior of immobilized anti-TPO-autoantibodies in immunoaffinity chromatography of this microsomal antigen was studied.

[A new property of known proteins: specific binding of thyroid hormones by human plasma apolipoproteins]. / Novoe svoistvo izvestnykh belkov: spetsificheskoe sviazyvanie tireoidnykh gormonov apolipoproteinami plazmy cheloveka.

The thyroid hormones--thyroxine (T4) and triiodothyronine (T3)--are capable of associating with human plasma high (HDL), low (LDL) and very low density lipoprotein particles (VLDL). Apolipoproteins (apo) act as the hormone-binding components of the lipoprotein particles. The thyroid hormone binding to apolipoproteins is a time-dependent, reversible, saturable and sensitive to specific inhibitors interaction with the structurally isolated site in the protein which is complementary to the ligand. The number of such sites per macromolecule varies from one (apoA-I) to three (apoB-100). Low affinities (Ka approximately 10(5)-10(6) M-1) for T4 are characteristic of apoA-II, apoA-IV, apoE and apoB-100, while apoA-I and its lipid complex, apoA-I-HDL display sufficiently high affinities for the hormone (Ka approximately 10(7)-10(8) M-1). The active site of apoA-I-HDL contains a chemical group interacting with the ionized phenolic ring hydroxy-group of T4, includes cavities capable of accommodating iodine atoms of the ligand molecule, has a hydrophobic nature and low stereospecificity and is located close to the surface of the protein globule in the N-terminal part of polypeptide chain conformationally associated with the polar surface monolayer of the lipid matrix. Three hormone-binding sites in apoB-100 are located in different regions of the polypeptide chain which are distant from each other and lie outside the sites of heparin and cellular LDL receptor binding. ApoB-100 and apoE stimulate T4 entry into fibroblasts, while apoA-I inhibits the interaction of T4 and T3 with human placental plasma membranes.

[Interaction of thyroid hormones with immunoglobulins isolated from human blood serum. II. Structural elements and localization of thyroxine-binding segment in the immunoglobulin M molecule]. / Vzaimodeistvie tiroidnykh gormonov s immunoglobulinami, vydelennymi iz syvorotki krovi cheloveka. II. Strukturnye élementy i lokalizatsiia tiroksinsviazyvaiushchego uchastka v molekule immunoglobulina M.

The structural characteristics and topography of the thyroxine T4-binding site on the human immunoglobulin M (IgM) molecule have been studied using affinity binding with T4 structural analogs blocking the formation of the T4-IgM complex with proteins displaying an affinity for individual structural components of IgM and proteolytic fragmentation followed by determination of the T4-binding activity of the isolated fragments. It has been found that IgM has a poor selectivity towards the binding of T4 structural analogs which is characteristic of transport proteins but not of autoantibodies. The T4-binding region of IgM lies outside the variable portion of the Fab-fragment and is apparently constituted by the Cmu 1-domain of the heavy chain and the constant part of the light chain linked via a disulphide bridge. The T4-binding site located within this region possesses no stereospecificity and contains structural elements complementary to the iodine atoms of the outer ring and the aliphatic chain of the iodothyronine molecule.

[Interaction of thyroid hormones with immunoglobulins isolated from human blood serum. I. Parameters of complex formation and the nature of the binding reaction]. / Vzaideistvie tireoidnykh gormonov s immunoglobulinami, vydelennymi iz syvorotki krovi cheloveka. I. Parametry kompleksoobrazovaniia i priroda reaktsii sviazyvaniia.

The kinetic and equilibrium characteristics of the interaction of thyroxine (T4) with immunoglobulins (Ig) A, G and M as well as with Bence-Jones proteins purified from human blood serum have been investigated. The formation of complexes between T4 and human immunoglobulins has been found to be time-dependent, reversible, saturable and sensitive to specific inhibitors. The L-chain (ae or lambda) is a component of the immunoglobulin molecular structure which appears to be essential and sufficient for the T4 binding. The covalent attachment of the H-chain can increase dramatically the affinity for the thyroid hormone (the mu-chain in IgM) or alter the sensitivity of the binding region to chemical agents and pH (the mu-chain in IgM, the gamma-chain in IgG). The experimental data suggest that the T4-binding IgM does not belong to a pathological type of proteins- anti-T4 autoantibodies-because: (i) the dependence of the T4 binding reaction on the physico-chemical conditions of the environment is typical of normal transport proteins; (ii) the prevalence of the T4-binding IgM in random individual serum samples from healthy subjects is 100%; (iii) the IgM-T4 complex differs structurally from the antigen-antibody complex, being unable to interact with the first complement component. The specific T4-binding properties of normal human serum immunoglobulins could remain so far unrecognized due to the inability of the conventional analytical methods to detect the weak manifestations of the T4-binding activity of these proteins in physiological fluids containing the endogenous inhibitor (Cl-), and/or the exogenous inhibitor (8-anilino-1-naphthalene sulphonic acid).

[Structural changes of lipid and thyroxine-binding protein zones of lipoprotein particles, containing human apolipoprotein A-1]. / Strukturnye izmeneniia lipidnoi tiroksinsviazyvaiushchei belkovoi zon lipoproteinovykh chastits, soderzhashchikh apolipoprotein A-1 cheloveka.

The relationship between temperature-induced structural changes in the lipid and protein thyroxine-binding zones of lipoprotein particles containing human apolipoprotein A-1 (apoA-1) as a sole protein component was studied using ESR with probes of two types--spin-labeled thyroxine and 5- or 16-doxylstearic acids. A structural rearrangement in the protein active site at 23-24 degrees was found. In the same temperature range, a dramatic change in the monolayer phospholipid ordered structure was observed. Based on these data, the localization of the apoA-1 thyroxine-binding site in the lipid phase as well as the possibility of regulation of the hormone binding process by changes in the protein-lipid interaction in lipoprotein particles are discussed.

[Simulating and inhibiting effect of individual blood serum transport proteins on thyroid hormone binding with human placental cell membrane]. / Stimuliruiushchee i ingibiruiushchee deistvie individual'nykh transportnykh belkov syvorotki krovi na sviazyvanie tireoidnykh gormonov s plazmaticheskimi membranami platsenty cheloveka.

Binding processes in in vitro systems modelling specific interactions between transport and receptor proteins, thyroid hormones of human placental tissue and the washing blood, have been studied. These systems included syncytiotrophoblast villous membranes, thyroxine (T4), triiodothyronine (T3) and iodothyronine-binding proteins: transthyretin, albumin, immunoglobulins (Ig) M and G, apolipoprotein A-I, and the T4-binding globulin purified from human retroplacental serum. All of the transport proteins at concentrations close to the Ka values of their complexes with thyroid hormones produced inhibitory effects on the binding of [125I]T3 or [125I]T4 to the thyroid hormone membrane receptor. The dependence of [125I]T4 membrane binding on IgM concentration in the system was characteristic of all proteins studied. In the case of T3 such dependence was unique for IgM, and included the phase of the IgM stimulatory effect (10(-11)-10(-9) M) and the phase of inhibition (10(-8)-10(-7) M). In the presence of 30 pM IgM, the concentration of the membrane T3-binding sites increased by 75% with a 2.2-fold decrease of the association constant (Ka). It was shown that IgM interacts specifically with two classes of binding sites on the plasma membranes with Ka(1) = 5.0 x 10(9) M-1, Bmax(1) = 34 fmol/mg of membrane protein and Ka(2) = 2.7 x 10(7) M-1, Bmax(2) = 2.0 pmol/mg of membrane protein. It is suggested that the stimulatory effect of IgM is caused by increases in the number of T3-binding sites on the placental villous membranes as a result of complex formation between IgM and its membrane receptor which displays an enhanced T3-binding activity.

[Thyroid hormone conjugates with rhodamine B as fluorescent ligands of human plasma transport proteins]. / Kon''iugaty tireoidnykh gormonov s rodaminom B kak fluorestsentnye ligandy transportnykh belkov plazmy krovi cheloveka.

Conjugates of thyroxine (T4) and triiodothyronine (T3) with rhodamine B in which the hormone and the fluorescent dye are linked via a thiourea bond have been synthesized. These conjugates possess an ability to inhibit in a competitive manner the binding of [125I]T4 to three protein preparations: T4-binding globulin (TBG), apolipoprotein A-I (ApoA-I), and high density lipoprotein particles (ApoA-I-HDL) isolated from human serum by T4-Sepharose 4B chromatography and further purified. The following values of association constants have been estimated: for the T4 derivative-3 x 10(7) M-1 (TBG), 4.1 x 10(5) M-1 (ApoA-I), and 4.2 x 10(5) M-1 (ApoA-I-HDL); for the T3 derivative-1.6 x 10(7) M-1 (TBG), 5.3 x 10(5) M-1 (ApoA-I), and 5.4 x 10(5) M-1 (ApoA-I-HDL). The binding of rhodamine B-labeled thyroid hormones to TBG or ApoA-I do not alter significantly the parameters of rhodamine B chromophore absorption and fluorescence. The interaction of the conjugates with ApoI-HDL leads to a significant enhancement of the absorption intensity and a 3 nm blue shift in the absorption maximum as well as to a 1.5-fold increase in the fluorescence band amplitude at 586 nm. Biological and fluorescent properties of T4 and T3 derivatives suggest that these compounds may be a useful tool in fluorescence studies of plasma binding protein-driven transport of thyroid hormones in model biological systems.

[Isolation, identification and properties of a new thyroxine-binding protein--apolipoprotein A-1 from human blood serum]. / Vydelenie, identifikatsiia i svoistva novogo tiroksinsviazyvaiushchegobelka--apolipoproteina A-1 syvorotki krovi cheloveka.

A protein having a molecular mass of about 25 kWa was isolated by thyroxin (T4)-Sepharose affinity chromatography from human blood serum; its properties were found to be distinct from those of known T4-binding proteins. Using immunodiffusion, radioimmunoassay, lipid analysis, differential precipitation and electrophoresis, it was shown that the isolated protein is a component of high density lipoprotein (HDL) particles and represents an apolipoprotein A-1 (apoA-1). Using cholate-Sepharose chromatography apoA-1 was separated from the lipid moiety and contaminant proteins, and apoA-1 was effectively isolated directly from the blood serum. Apo-A-1-HDL and apoA-1 obtained by affinity chromatography as well as the HDL3 fraction isolated by a standard ultracentrifugation technique, all displayed a T4-binding activity, the affinities for the hormones being of the same order of magnitude. The T4 interaction with these preparations induced difference UV-absorption signals, altered the characteristics of apoA-1 intrinsic fluorescence without affecting the circular dichroism of the protein-hormone system. The binding of spin-labelled T4 to apo-1, apoA-1-HDL or HDI3 caused substantial changes in the shape of the ESR spectrum and an increase in the apparent rotational correlation time. The mobility of the radical fragment of spin-labelled T4 depended on the composition and properties of the protein preparation. The electron spectroscopy data suggest that the T4-HDL interaction occurs via specific mechanisms and that the molecular structures of the complexes formed thereby are not characteristic of other known T4-binding proteins.

[Pregnancy-associated molecular variation of thyroxine-binding globulin in health and in various diseases of man]. / Sviazannaia s beremennost'iu molekuliarnaia raznovidnost' tiroksinsviazyvaiushchego globulina v norme i pri nekotorykh zabolevaniiakh u cheloveka.

A molecular variant of human serum thyroxine-binding globulin (TBG), containing only triantennary oligosaccharide chains and having a more prolonged in vivo survival (TBG-1), was first detected in normal pregnancy and then in the postpartum period. Serum TBG-1 was measured in normal and in some pathological conditions associated with normal or increased TBG biosynthesis using a combination of Con A-Sepharose 4B microcolumn affinity chromatography and a highly sensitive TBG radioimmunoassay. TBG-1 was shown to be present in the sera of healthy subjects (0.21 +/- 0.04 micrograms/ml, n = 60; 1.15% of total TBG). The proportion of TBG-1 in total serum TBG was significantly increased (up to 9.5%) in conditions with serum TBG excess (cancer, hypothyroidism and liver diseases). It has been assumed that a selective enhancement of biosynthesis of the TBG molecular variant with a prolonged half-life in the circulation during the posttranslational modification of the polypeptide chain is a response to the body requirements in a high TBG concentration.