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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.

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.

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 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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

The kinetic and equilibrium characteristics of interaction of thyroxine (T4) and its structural analogs with a high density lipoprotein (HDL) fraction isolated from human serum by T4-Sepharose affinity chromatography and containing apolipoprotein A-I (apo A-I) as a sole protein component, were studied. The binding of [125I]T4 to apo A-I-HDL reached a maximum after 40 min and did not change during the next 80 min of incubation at 0 degrees--22 degrees C. Dissociation of [125I]T4 induced by the addition of excess unlabeled T4 to the complex solution proceeded more intensely on a time scale at 0--2 degrees C than at 22 degrees C. Incubation of apo A-I-HDL with increasing concentrations of T4 showed that the binding is saturable. The data analysis using different computer programs revealed the presence in apo A-I-HDL of a single class of binding sites with K alpha = (4.0 +/- 2.1).10(-7) M- and Bmax = 1.7 +/- 0.8 nmol T4/mg of protein. Naturally occurring iodothyronines, their analogs and D-isomers of thyroid hormones competed with [125I]T4 for the binding sites on apo A-I-HDL with the following inhibitory potencies: L-T4 = D-T4 greater than or equal to 3,3',5-triiodo-L-thyronine = 3,3',5-triiodo-D-thyronine greater than 3,5-diiodo-L-thyronine = 3,3',5- triiodothyroacetic acid greater than 3,3',5-triiodothyropropionic acid greater than or equal to 3,5-diiodo-L-thyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)

The serum concentrations of the total thyroxine-binding globulin (TBGt), its pregnancy-associated molecular variant (TBG-1), thyroxine, progesterone, estradiol, and prolactin were measured by radioimmunoassay in the course of the postpartum period (0-40 days) in women. High serum TBG-1 concentrations (an average of 36% of the level observed at the time of delivery) were unexpectedly detected in the late postpartum period (days 36-40) when the concentrations of TBGt, thyroxine, estradiol and progesterone were within normal. The presence of TBG-1 in the maternal blood for a long time after delivery may be caused by estrogen-dependent synthesis and secretion rather than by its slow clearance from the blood.

Kinetic and equilibrium characteristics of the interaction of triiodothyronine (T3) and its structural analogues with plasmatic membranes of human placental syncytiotrophoblast were studied. The specific binding was temperature-dependent, saturable, reversible, selective and stereospecific. The following values of the binding parameters were obtained: association rate constant--1.0 x 10(10) M-1min-1, dissociation rate constant (fast phase)--2.4 x 10(-1) min-1, half-life time of the complex--2.9 min, equilibrium association constants for high- and low-affinity sites--2.0 x 10(10) M-1 and 4.8 x 10(6) M-1, respectively, concentrations of the corresponding sites--12.5 fmol and 36.0 pmol per mg of membrane protein, respectively. The affinity for membranes decreased in the following order: L = T3 greater than L = T4 greater than 3,3,5-triiodothyroacetic acid greater than 3,3,5-triiodothyropropionic acid greater than D = T3 greater than D = T4 greater than 3,5-diiodo-L-tyrosine.

The affinity matrix prepared by the attachment of L-thyroxine (T4) to epichlorohydrine-activated Sepharose 4B biospecifically absorbs the T4-binding globulin (TBG), 25K and 80/27K proteins, immunoglobulin G (IgG) and albumin (HSA) from human normal and retroplacental sera. The absorbed protein patterns were shown to depend on the immobilized T4 concentration, pH, temperature and incubation time. The potent eluents desorbing 85-100% of the protein are 1 mM NaOH, 3 M NH4SCN, 10(-5) M T4 or 3 mM 8-anilinonaphthalene-1-sulfonic acid (ANS) for TBG; NaOH, NH4SCN, 3 mM MgCl2 or 12mM sodium cholate for 25K protein and HSA; NaOH, NH4SCN or MgCl2 for the 80/27K and 25K proteins and IgG. Moreover, T4 desorbs small amounts (6-8%) of the 80/27K and 25K proteins, while sodium cholate elutes about 6% of TBG. The eluted from T4-Sepharose 4B and further purified TBG, 25K and 80/27K proteins display different [125I]T4-binding activities within the pH range from 2 to 9 and differ by their resistance to thermal inactivation at 50-80 degrees C. Double radial immunodiffusion analysis with the use of antisera to TBG, 25K, 80/27K, HSA and IgG demonstrated that the proteins share no common antigenic determinants. It was concluded that the novel 25K and 80/27K proteins represent endogenous components of the human blood thyroid hormone-binding protein system rather than fragments or aggregates of the known T4-binding proteins.

Two molecular variants, of thyroxin-binding globulin (TBG), TBG-1 and TBG-2, were obtained from human retroplacental blood by fractionation of pure TBG on concanavalin A-Sepharose. It was found that both variants are immunologically identical, have similar molecular weights, amino acid composition and spectral properties, and possess the same affinity for thyroid hormones. However, TBG-1 and TBG-2 differed in charge upon isoelectrofocusing and had different monosaccharide composition. The existence of two molecular variants of TBG in pregnancy is probably due to the peculiarities of the polypeptide chain glycosylation during TBG biosynthesis.

Total thyroxine--binding globulin (TBGt) and its pregnancy-associated molecular variant (TBG-1) were detected by radioimmunoassay in human amniotic fluid collected at the time of delivery. TBGt purified from amniotic fluid by affinity chromatography and hydroxylapatite chromatography, displayed electrophoretic properties, immunoreactivity, a molecular mass, affinity for thyroxine and TBG-1 content identical to those of pure TBGt from human pregnancy serum. The concentrations of TBGt and TBG-1 in maternal venous blood serum were 49 +/- 7 mg/ml and 3.8 +/- 1.3 mg/ml (n = 23), respectively, and those in amniotic fluid were 2.0 +/- 0.5 mg/ml and 0.19 +/- 0.12 mg/ml (n = 30), respectively. The analysis of paired samples showed that the serum and amniotic fluid TBGt levels closely correlated (r = 0.91), whereas the correlation between the respective TBG-1 levels was poor (r = 0.63). The TBG-1/TBGt ratio in amniotic fluid was 20% higher than that in serum. These findings suggested a maternal origin of the most of the amniotic fluid TBG which may possibly enter amniotic fluid by passive diffusion through fetal membranes. An alternative mechanism can exist for TBG-1 transfer from maternal serum to amniotic fluid. It corresponds to a special role that TBG-1 may play in the fetoplacental system.

Using spectroscopic, electrophoretic and microcalorimetric techniques, the changes in the spatial structure of human thyroxine-binding globulin (TBG) induced by exposure of protein solutions to high temperatures (45-90 degrees C) and low pH (2.5-6.0) were studied. Simultaneously the biological activity and immunoreactivity of TBG samples were measured. The structural changes were manifested at 52 degrees C or at pH 4.0 and were then aggravated with a rise in temperature or a decrease of pH. The circular dichroism spectra showed that the molecular ellipticity had a maximum decrease (by 10%) at 218-222 nm. In fluorescence spectra excitable at 280 nm the band half-width increased by 4-6 nm; their intensity decreased by 30-40%, whereas the position of the maxima did not change significantly. After addition of an equimolar amount of thyroxine to inactivated TBG the protein fluorescence was quenched by 25-40%. The electrophoregrams of treated preparations contained additional protein bands possessing no biological activity, whose mobility was less than that of native TBG. Microcalorimetric assays of native TBG revealed a thermoabsorption peak with a maximum at 62.5 degrees C and a half-width of 7.1 degrees C. The thermodynamic parameters of melting of TBG spatial structure were consistent with a model of a two-domain structure of the molecule. The biological activity and immunoreactivity of TBG showed a coordinated decrease with a rise in the degree of protein denaturation, However, the formation of TBG complex with antibodies did not screen the thyroxine-binding center of TBG and did not alter its affinity. Possible mechanisms of structural transition of TBG and its effect on the biological properties of TBG are discussed.

A new technique for purification of the human sex hormone-binding globulin (SHBG) is described. This technique includes affinity chromatography of blood serum on cortisol-Sepharose, (NH4)2SO4 fractionation, gel filtration on a Bio-Gel P-300 column and chromatography on a concanavalin A-Sepharose 4B column. From 21 of retroplacental serum 10 mg of pure SHBG (25% yield) has been obtained. Upon gel filtration SHBG behaved as a biopolymer with Mr of 120,000. The molecular weight of SHBG as determined by electrophoresis was shown to be equal to 50,000. SHBG has a sedimentation constant of s20, w of 4.7S, pI of 5.75, extinction coefficient A1%(280,1cm) = 10,5 and association constants of 4.5 X 10(8) and 3.5 X 10(6) M-1 for 5 alpha-dihydrotestosterone and cortisol, respectively. The amino acid and carbohydrate contents of SHBG were determined.

The concentrations of the molecular variant of the thyroxine-binding globulin (TBG-1) and total TBG were measured in normal human sera (male and female), venous blood sera at various stages of normal pregnancy as well as in matched samples of retroplacental and venous sera of mothers and in cord sera of their newborns. It was shown that TBG-1 appeared on the 6th-11th week of pregnancy and its level increased up to the delivery. A TBG-1 portion in the total TBG content increased from 2.0% in the 3rd month of pregnancy to 8.8% in the last month. After delivery TBG-1 and TBG levels decreased on the first day from 3.82 +/- 0.52 micrograms/ml to 2.52 +/- 0.86 micrograms/ml (P less than 0.01) and from 43.3 +/- 7.2 micrograms/ml to 37.8 +/- 9.6 micrograms/ml (P less than 0.01), respectively. However on the 5th day a decrease in TBG-1 and TBG concentrations were not observed. TBG-1 was undetectable in normal adult and cord sera. TBG-1 concentrations in matched retroplacental and venous sera at term were similar. These results suggested that TBG-1 was related to pregnancy-associated proteins rather than to proteins of fetal or placental origin.

Based on the new data concerning the multicomponent system of thyroxine-binding proteins in human plasma, some methodological aspects of isolation and purification of thyroxine-binding globulin (TBG) were examined, and a simple two-step procedure for TBG purification was developed. Normal human blood serum, retroplacental serum and amniotic fluid were used as TBG sources. The procedure includes affinity chromatography and adsorption chromatography on a hydroxyapatite column. A biospecific adsorbent was synthesized by stepwise binding of epichlorohydrin and thyroxine to Sepharose. The yield of pure TBG varied from 60 to 80%, depending on the TBG source used. The properties of TBG preparations from retroplacental serum and amniotic fluid were identical; both preparations contained a pregnancy-associated molecular variant, TBG-1. Two novel serum thyroxine-binding proteins were detected, isolated and partly characterized.

Human retroplacental blood plasma proteins with affinity for cortisol were isolated by biospecific chromatography and identified by electrophoretic and immunochemical methods as alpha1- and beta1-globulins and IgG. IgM and IgA immunoglobulins. A high specific affinity for cortisol (Kas = 1,5 . 10(8) M-1 at 23 degrees C) and progesterone (Kas = 2,0 . 10(8) M-1 at 23 degrees C) was observed only for alpha-globulin; other proteins had a low affinity for cortisol. The molecular weight of alpha1-globulin (transcortin) was found to be 50,000-55,000. The amino acid and monosaccharide compositions of this glycoprotein were studied. Its N-terminal and C-terminal amino acid sequences are: Met-Asx-Pro-Asx-Ala- and (Val, Gln)-Leu, respectively. It was concluded that under normal physiological conditions and during pregnancy transcortin is the only specific corticosteroid-binding plasma protein. A complete removal of bound cortisol from the protein mixture and subsequent hydroxylapatite chromatography resulted in homogeneous transcortin retaining more than 90% of its binding capacity. The formation of the transcortin-steroid complex and its complete dissociation are accompanied by conformational changes of the protein globule. Significant changes of the spectral properties of the tryptophane residue of protein and the steroid delta4-3-keto group are indicative of the possibility of their direct interaction.