Comparative study of conformation properties of Fc-fragments of human immunoglobulin G subclasses using 1H-NMR

Conformational properties of the Fc- and pFc'-fragments of human myeloma immunoglobulins G of the first and third subclasses were studied by 1H-NMR method (270 and 400 MHz). It was found that the globular structures (domains) of the Fc-fragments of IgG1 and IgG3 in solution are characterized by high segmental mobility, and have no significant differences in their spatial arrangement. Comparative analysis of the spectra obtained at different temperatures (30-70 degrees C) revealed that the Fc-fragment of IgG3 has a more heat-stable conformation than the Fc of IgG1. The intramolecular mobility of the Fc-fragment increased upon lowering the pH. The partial assignment of the signals in the NMR spectra of the Fc-fragments of immunoglobulins G1 and G3 was carried out, and the pKa values for histidines of the pFc'-fragment of IgG1 were determined.     

Comparative conformational studies on the tryptic digestion fragments of human immunoglobulins M and G

IgM¹ immunoglobulins were cleaved into Fabμ and (Fc)5μ fragments by tryptic digestion. Comparative circular dichroism studies with the corresponding IgG fragments show that the Fab portions of IgG and IgM proteins have very similar CD spectral features, although the same is not true for their Fc fragments. These studies indicate the presence of higher amount of beta-structured regions in Fcμ than in Fcγ. Also, there are considerable differences in their pH-dependent structural transitions as measured by CD spectral changes. The conformational differences between IgG and IgM immunoglobulins are more pronounced in their Fc portions, which carry out class specific biological functions, rather than in Fab portions, which contain antigen combining sites.     

The structural-functional properties of light and dark hepatoma cells

Morphofunctional analysis of the light and dark solid hepatoma cells of mouse line 22a C3HA and culture of human hepatoma has shown that in both hepatomas the dark cells when compared with the light ones possess increased electronic density of hyaloplasm, and besides the dark cells of solid tumor line have a well-developed endoplasmic reticulum and numerous mitochondria. Both types of cells are able to incorporate labelled RNA and DNA precursors and also to divide themselves mitotically.     

Study of the antigenic relationship between animal and human immunoglobulins using antisera to human immunoglobulins]

Cross-reactivity of monospecific antisera to human immunoglobulins with animal sera of 10 species was studied by immunoelectrophoresis and radial immunodiffusion. Antisera to IgG were shown to reveal IgG of all the species studied, antisera to IgM and especially to IgA cross reacted less extensively. The greatest number of cross reactions were given by the antisera obtained as a result of hyperimmunization. Hyperimmune monospecific antisera to human IgG, IgA, and IgM can be used for the identification of animal immunoglobulins during their isolation from the sera and for their quantitation by radial immunodiffusion.     

Synthesis of immunopoietins--new biologically active low molecular weight fragments of immunoglobulins G, M, E and A

Low-molecular fragments of immunoglobulins IgG-(245-349) (Glu-Pro-Gln-Val-Tyr), IgM-(451-455) (Arg-Pro-Asp-Val-Tyr), IgA-(347-351) (Arg-Pro-Glu-Val-His), and IgE-(430-435) (Ala-Ala-Pro-Glu-Val-Tyr), potentially active immunoregulators of a novel type, have been synthesised by classical methods of peptide chemistry. This group of compounds, which in biological effects are similar to thymopoietin, was given the name of immunopoietins.     

Analysis of highly tritium-labeled alanine using 3H- and 1H-NMR

A method has been developed for the analysis of deuterium oxide solutions of tritiated alanine eight-component isotopic mixtures by using high resolution 3H and 1H NMR spectra at frequencies 266.8 and 500.13 MHc, respectively. Approaches have been worked out for the determination of qualitative composition of the mixtures and spectral parameters.     

Syntheses of branched trinucleotides and their conformational analysis by 1H-NMR spectroscopy

Unambiguous chemical synthesis of branched ribonucleotides, which are products of splicing reactions in eukaryotic cells, and their analogues are reported, subsequently, their secondary structures have been determined by a 270 MHz 1H-NMR spectroscopy.     

Structural analysis of carbohydrate chains of normal and myeloma immunoglobulins G using HPLC

A comparative analysis of carbohydrate chains from human normal IgG and myeloma IgG (subclass 4) was carried out using HPLC. It is shown that both IgGs contain the same carbohydrate chains (biantennary and bisected) but the relative amount of bisected and incomplete chains (with fewer terminal galactose residues) is higher in myeloma IgG4. Two earlier unknown minor oligosaccharides were isolated from normal IgG and structurally studied by means of the partial hydrolysis and the Smith degradation.     

Independent folding of autolytic fragments of thermolysin and their domain-like properties

High molecular weight autolysis products from thermolysin have been isolated and identified. The primary fragments correspond to residues 1 to 187-204 (21kD) and residues 187-204 to 316 (12kD), respectively. The fragments are both capable of independent refolding upon removal of denaturant. On the basis of these results, we suggest that the first step in the unfolding pathway of thermolysin involves unfolding of an interdomain region and domain separation. Bound calcium ions at sites 1, 2 and 4 play a major role in protecting the protein against both autolysis and unfolding, probably by stabilizing the interdomain region and enhancing domain-domain interactions.     

The structural-functional organization of G-proteins and their bound receptors]

Recent data on the structure, function and regulation of different types of GTF-binding proteins (G-proteins), playing the key role in transmembrane signalling, and of G-protein-coupled receptors are summarized. Possible mechanisms involved in the receptor-G-protein coupling and G-protein-membrane connection are discussed.     

Study of peptide degradation in the serum by 1H-NMR

Enzymatic degradation of hexapeptide Tyr-D-Ala-Gly-Phe-Leu-Arg in human serum has been investigated by 1H-NMR spectroscopy, and its pathways are suggested. Its final degradation products, tetra- and pentapeptide, are shown to be stable in human serum for several hours. The NMR spectroscopy application for pharmacokinetic studies is substantiated.     

Mechanistic study of the urocanase reaction using deuterated substrates and 1H-NMR spectroscopy

1. Samples of (alpha-2H1, 5-2H1) and (alpha-2H1, beta-2H1) urocanic acid were prepared by a combination of chemical and enzymic methods. 2. The enzymic conversion of unlabelled urocanate was followed by 1H-NMR spectroscopy at 500 MHz in deuterium oxide. It was found (a) that urocanase promotes the exchange of the 5-hydrogen atom of the substrate faster than it catalyses the overall reaction, (b) that the product is an equilibrium mixture of racemic beta-(5-oxoimidazol-4-yl)propionate and beta-(5-hydroxyimidazol-4-yl)propionate and (c) that beta-(5-oxoimidazol-4-yl)-propionate is spontaneously hydrolysed under physiological conditions to N-formylisoglutamine. The rate of this hydrolysis is considerably diminished at +8 degrees C. 3. It was shown by ultraviolet and 1H-NMR spectroscopic measurements that beta-(5-hydroxyimidazol-4-yl)-propionate (gamma max approximately equal to 234 nm) exists in protonated from at low pH (less than 1) whereas pH (approximately equal to 7.5) it exists in equilibrium with beta-(5-oxoimidazol-4-yl)propionate (gamma max approximately equal to 269 nm). 4. (alpha-2H1, beta-2H1)Urocanate was reacted with urocanase in deuterium oxide. 1H-NMR spectroscopy at 500 MHz showed a slight incorporation of protium into the side-chain of the product. The incorporated protium corresponded roughly to the protium contamination of the solvent and was equally distributed between the alpha and beta positions. No transfer of the 5-hydrogen atom to the side-chain was detected. 5. Kinetic deuterium isotope effects of between 2 and 3 were measured when the urocanase reaction was conducted in deuterium oxide at different p2H values. 6. Implications of these findings for the mechanism of action of urocanase are discussed.     

Role of G0- and G1-splenocytes and antigen-binding lymphocytes in the production of antigen-dependent nonspecific immunoglobulins

Immunization of irradiated and syngeneic splenocyte-treated CBA mice with bovine red blood cells stimulated the formation of both antigen-producing cells (APC) and antigen-dependent nonspecific immunoglobulin-producing cells (NIGPC). The injection of G0-cell-enriched, instead of normal, splenocytes (together with bovine RBC) to irradiated mice reduced by half the production of antigen-dependent NIGPC. Thus, it is evident, that some of them are formed from preexisting blast cells (G1). An additional removal of antigen-binding cells (ABC) from G0 lymphocyte population produced a still greater reduction in NIGPC formation (the increment was 92.3% lower than in the control). It is concluded that antigen-dependent NIGPC are formed both due to specific antigen-stimulation of G0 cells aid to nonspecific stimulation of blast cells with factors produced by antigen-stimulated T-cells.     

Electrical detection of human immunoglobulins G from human serum using a microbiosensor

A biosensor for the electrical detection of human antibodies from serum has been fabricated and experimentally demonstrated. The device is based on the immobilization of proteins used as probes between a set of microelectrodes. Incubation with diluted human serum was followed by incubation with anti-human secondary antibodies labeled with gold nanoparticles (GNPs) and then precipitation of silver on the nanoparticles. The output of the device was defined as the percentage of short-circuited microelectrodes after silver deposition independently of the gap conductance. Two model probes were studied: protein A and goat antibodies. The effects of the microgap spacing (5, 10, 15 or 20 microm) and the duration of the silver treatment were examined. The data obtained showed that a large spacing (20 microm) led to poor sensitivity. Alternately, 5 microm gaps led to high sensitivity and saturation of the signal. Interestingly, 10-15 microm gaps enabled a non-saturated and distinct signal for both probes that was correlated with the GNP density between the microgaps as determined by atomic force microscopy. Different capture efficiencies could be easily distinguished. The biosensor described here is easy to use and thus can be applied to real detection experiments.     

Improved quantification from 1H-NMR spectra using reduced repetition times

A method of choosing the correction factor based on Bloch equation for the quantitative estimation of metabolite from ¹H NMR spectra recorded with reduced recycle delay is prescribed. The procedure reduces the experimental time without substantially compromising the accuracy of quantitative estimation. It is based on choosing the correction factors, which depend on T₁ and T₂ of the metabolite and recycle delay used for recording the spectra. It is validated by studying a mixture of amino acids with known concentration of constituents and human serum sample and it provides accuracy of quantitative estimation to 95–96%.     

Calcium-binding properties and molecular organization of bradykinin A solution 1H-NMR study.

The NMR features of bradykinin were investigated in dimethylsulfoxide containing 1% water. The temperature dependence of chemical shifts and ROESY maps were monitored for the major species where all X-Pro bonds are trans. The occurrence of a head-to-tail ionic interaction and intramolecular hydrogen bonds stabilizing a pseudo cyclic arrangement was inferred, a beta turn at the C-terminus being the main feature of the secondary structure. Calcium was shown to bind to the peptide with a dissociation constant Kd = 2.8 + 0.2 mm. 2Pro and 3Pro carbonyls, as well as the 9Arg carboxyl, were assigned as the metal-binding sites. A molecular model of the 1 : 1 metal-complex was obtained. In light of conformational changes experienced by the peptide upon interaction with calcium, a role for the metal was hypothesized in the process of conformational selection from the free to the receptor-bound state of bradykinin.