chloramine-t and 1-3-4-6-tetrachloro-3-alpha-6-alpha-diphenylglycoluril

Topics: Animals; Cell Line, Tumor; Cell Membrane; Chloramines; Halogenation; History, 20th Century; Immunoglobulin G; Iodine; Mice; Mice, Inbred BALB C; Proteins; Solubility; Tosyl Compounds; Urea

Topics: Chemokine CCL4; Chemokines; Chloramines; Chromatography, Gel; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Interleukin-8; Iodine Radioisotopes; Isotope Labeling; Lactoperoxidase; Macrophage Inflammatory Proteins; Protein Binding; Receptors, Chemokine; Tosyl Compounds; Urea

Several methods for the iodination of recombinant v-H-ras protein were compared. The Iodobead method gave greatest incorporation of radioactivity with minimal modification of the ras protein. Upon treatment of the ras protein with [125I] Nal and an Iodobead, radioactivity was initially incorporated into a 22 kDa species with a pl of 5.2, then predominantly into a 23 kDa species with a pl of 5.4. The specific activity of [125I]ras was 6 x 10(6) cpm/pmol total ras protein. Iondination did not alter the biological activity of the ras protein as judged by its ability to bind GTP gamma S and induce maturation of Xenopus laevis oocytes. It is concluded that while iodination alters the apparent molecular weight and pI of ras, presumably by the oxidation of one or more classes of amino acids, this does not affect the biological function of the protein. The ras protein, radioactively-labelled with iodine using the Iodobead method, should be suitable for studies of protein-protein interactions involving ras. Treatment of iodinated ras with the chemical cross-linking agent disuccinimidyl suberate revealed the presence of several minor high molecular weight protein species. This result shows that, in a dilute solution of purified ras protein, the monomeric form is in equilibrium with small amounts of polymeric forms.

Topics: Animals; Blotting, Western; Chloramines; Cross-Linking Reagents; Electrophoresis, Gel, Two-Dimensional; Female; Guanosine 5'-O-(3-Thiotriphosphate); Iodine Radioisotopes; Isoelectric Point; Isotope Labeling; Lactoperoxidase; Methods; Microspheres; Molecular Weight; Oncogene Protein p21(ras); Oogenesis; Polymers; Recombinant Fusion Proteins; Sodium Hypochlorite; Succinimides; Tosyl Compounds; Urea; Xenopus laevis

Six different procedures for radioiodination of mouse epidermal growth factor (EGF) all resulted in a heterogeneous 125I-labeled EGF preparation, as analyzed by reversed-phase HPLC. EGF preparations that had been iodinated with Chloramine T, lodogen, or lodo-beads were found mainly to consist of oxidized 125I-labeled EGF moieties. In contrast, the heterogeneous 125I-labeled EGF preparations obtained by using iodine monochloride, Protag-125, or lactoperoxidase-glucose oxidase-coupled beads (Enzymobeads) contained insignificant amounts of oxidized EGF entities. Ligand equivalence analysis (LEA) of distinct HPLC column fractions, obtained after preparative separation of Chloramine T-125I-labeled EGF, showed that the receptor-binding affinity of the tracer in all subfractions was less than the affinity of unlabeled EGF. This implies that HPLC purification of these 125I-labeled EGF preparations does not yield 125I-labeled EGF preparations with ligand equivalence. However, all but one HPLC column fraction of Enzymobeads-125I-labeled EGF showed ligand equivalence. Despite the small amount of the nonequivalent component in the Enzymobeads-labeled tracer, the nonchromatographed 125I-labeled EGF preparation showed ligand equivalence. No significant differences were observed in the maximal binding capacity of the different 125I-labeled EGF preparations.

Topics: Animals; Chloramines; Chromatography, High Pressure Liquid; Epidermal Growth Factor; Hydrogen Peroxide; Iodine Radioisotopes; Isotope Labeling; Mice; Microspheres; Oxidation-Reduction; Tosyl Compounds; Urea

Following optimization of the reaction conditions, e.g. concentration of oxidizing agents, reaction time, volume of reaction mixture, and pH, chloramine T and the new iodination reagent, Iodogen, were compared for their effectiveness in radioiodination of insulin, glucagon, human growth hormone (hGH), and rabbit anti-mouse IgG. The radioactive peptide hormones prepared were analyzed for the presence of aggregate and breakdown products by polyacrylamide gel electrophoresis (PAGE) at pH 8.9, the rabbit anti-mouse IgG was tested for the presence of low molecular weight damage products by gel filtration on Sephadex G-50. The results demonstrate that with respect to iodine incorporation, specific activity, and immunological reactivity either method can be used to prepare under carefully controlled conditions a wide range of tracers with high specific activity at minimal oxidation damage. These tracers are shown to be highly suitable in radioimmunoassays after previous purification by PAGE and gel filtration, respectively.

Topics: Chloramines; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Glucagon; Growth Hormone; Hydrocarbons, Iodinated; Immunoglobulin G; In Vitro Techniques; Insulin; Iodine Radioisotopes; Isotope Labeling; Tosyl Compounds; Urea

Radioiodination provides an extremely sensitive method for the detection of low levels of proteins. In the development of a sensitive radioimmunoassay for human alpha-lactalbumin (alpha-LA), the protein was labeled to high specific activity (approaching 2000 Ci/mmol) with lactoperoxidase, chloramine-T, and Iodogen. Despite high specific activities of the labeled protein by each method, there was a considerable difference in their binding affinity with monoclonal anti-human alpha-LA antibodies due to varying degrees of protein damage. Iodination of human alpha-LA with Iodogen resulted in labels of the highest specific activity and immunoreactivity with the monoclonal antibodies used.

Topics: Antibodies, Monoclonal; Chloramines; Humans; Indicators and Reagents; Iodine Radioisotopes; Isotope Labeling; Lactalbumin; Lactoperoxidase; Precipitin Tests; Radioimmunoassay; Tosyl Compounds; Urea

Topics: Animals; Binding Sites, Antibody; Chloramines; Chromatography, High Pressure Liquid; Enzymes, Immobilized; Humans; Iodine Radioisotopes; Somatostatin; Spectrophotometry, Ultraviolet; Tosyl Compounds; Urea

Insulin receptors from human placenta have been labeled by using an oxidative iodination procedure (iodogen-mediated or chloramine-T-mediated), Bolton-Hunter reagent or [3H]acetic anhydride. The oxidative iodination procedure reduces the affinity for 131I-insulin and the receptor protein becomes fragmented into smaller pieces with an s20,w value of 5-6. However, treatment with Bolton-Hunter reagent or [3H]acetic anhydride does not alter the Kd of 131I-insulin binding and the s20,w value remains unchanged with respect to the native receptor. It is proposed that for labeling multisubunit sulfhydryl-linked protein drastic oxidative iodination procedures should be avoided.

Topics: Acetic Anhydrides; Acetylation; Chloramines; Humans; Insulin; Iodine Radioisotopes; Isotope Labeling; Kinetics; Oxidation-Reduction; Placenta; Receptor, Insulin; Succinimides; Tosyl Compounds; Urea

For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.

Topics: Chloramines; Hydrogen Peroxide; Indicators and Reagents; Iodine Radioisotopes; Isotope Labeling; Proteins; Radiochemistry; Succinimides; Tosyl Compounds; Urea

Radioiodination of the two tyrosine residues (Tyr-99 and Tyr-138) of ox testis calmodulin was performed using several methods, and studied through the specific activity, and the [125I]iodoamino acid analysis of the radiolabeled calmodulins. Hydrolysis by thrombin of 125I-calmodulin labeled by the lactoperoxidase method and subsequent isolation of peptides TM1 and TM2 by gel electrophoresis showed preferential labeling by 125I of Tyr-99 (TM1) over Tyr-138 (TM2). Analysis of [125I]iodoamino acids of radiolabeled TM1, TM2 and calmodulin demonstrated that [125I]monoiodotyrosine was predominant, the remainder being [125I]diiodotyrosine. Radioiodination of wheat germ calmodulin, which contains a single tyrosine residue (Tyr-139), showed that only TM2 was labeled by 125I on the Tyr-139 residue and also on the His-108 residue (radiolabeled monoiodotyrosine, diiodotyrosine and monoiodohistidine being present).

Topics: Animals; Calmodulin; Cattle; Chloramines; Iodine Radioisotopes; Isotope Labeling; Lactoperoxidase; Male; Monoiodotyrosine; Peptide Fragments; Plants; Testis; Thrombin; Tosyl Compounds; Triticum; Tyrosine; Urea

Synthetic calcitonin (CT) and Tyr(0)-katacalcin (tKT) were radioiodinated with Iodogen to high specific activity and with high yields. The products of the iodination procedure were chromatographed on Sephadex G-25 to remove unreacted iodide and then separated by chromatofocussing on PBE 94 (pH 9.6-6.0 for CT and pH 7.4-4.0 for tKT). Clear separation between uniodinated peptides and their mono- and di-iodinated derivatives was achieved with specific activities of 1900 and 3800 Ci/mmol for the respective mono- and di-iodinated peptides. Yields were up to 36 and 24% of mono- and di-iodinated CT and 41 and 29% for mono- and di-iodinated tKT. Our results show that Iodogen provides an effective and gentle way to iodinate peptides with high efficiency. Chromatofocussing is a simple, inexpensive and instrumentally undemanding method that can be performed without specialized chromatographic equipment and that should be applicable to a variety of different tracer preparations.

Topics: Calcitonin; Chloramines; Chromatography, Gel; Iodine; Iodophors; Lactoperoxidase; Peptide Fragments; Peptides; Tosyl Compounds; Urea

The presence of polymer(s) of radioiodinated bungarotoxin (Bgt) in preparations iodinated with chloramine-T or with iodogen, was investigated by chromatography on Sephadex-G50. In addition to a monomeric peak (P2), chloramine-T preparations showed a high content of polymeric forms (16-43%) eluting in the void volume (P1), present only to the extent of 1-3% in iodogen preparations. In purified [125I]Bgt prepared by the iodogen method, the content of P1 increased on treatment with chloramine-T, in the absence of radioiodine. The nonspecific binding of [125I]Bgt to DE-81 filter discs was high in the case of chloramine-T preparations and proportional to the content of P1 in each batch. Purified P1 (polymer) but not P2 (monomer) showed a concentration-dependent high degree of binding to DE-81 discs.

Topics: Bungarotoxins; Chemical Phenomena; Chemistry; Chloramines; Chromatography, Ion Exchange; Dextrans; Iodine Radioisotopes; Polymers; Tosyl Compounds; Urea

The relative reactivity of the tyrosine side chains in the proteins of skeletal muscle myofibrils was determined using iodination techniques. The destruction of ATPase activity of myofibrils and myosin by lactoperoxidase and chloramine-T iodination could be prevented by the attachment of cysteamine to the sulphydryl groups prior to the iodination reaction and subsequent regeneration with thioglycolate or dithiothreitol. Iodination using 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril did not require cysteamine treatment for retention of full enzymatic activity. The specific activity of the different proteins varied markedly with desmin, troponin-T, and tropomyosin having the highest labelling with all three iodination procedures. In contrast the myosin light chains had low specific activity when labelled in myofibrils or intact myosin. The isolated light chains, however, were much more highly iodinated. It appears that iodination may be a useful technique for examining protein-protein interactions in the myofibril.

Topics: Adenosine Triphosphatases; Animals; Autoradiography; Chemical Phenomena; Chemistry; Chloramines; Cysteamine; Iodine; Lactoperoxidase; Muscle Proteins; Myofibrils; Myosins; Rabbits; Tosyl Compounds; Tyrosine; Urea

A radioimmunoassay was developed for the direct quantification of aldolase B in human serum and tissues. The method is a double antibody radioimmunoassay technique using radioiodinated aldolase B homopolymer as ligand, chicken antibodies to aldolase B and rabbit antibodies to chicken IgG. This radioimmunoassay was shown to be specific for the aldolase B subunit, with no cross-reactivity with either human aldolase A subunit or homopolymeric human aldolase C (C4). The lowest measurable amount by this method was 2 ng/ml. Aldolase B is predominantly found in normal liver tissue, with relatively high aldolase B levels also observed in kidney. Aldolase B levels in the serum obtained from 11 normal subjects ranged from 23 to 38 ng/ml, with a mean of 28.5 +/- 9.2 (S.D.) ng/ml. Almost all of patients with hepatitis had serum aldolase B levels greater than 30 ng/ml. In cancer patients, serum aldolase B was slightly elevated in patients with metastatic liver cancer and primary lever cell carcinoma, whereas no elevation of serum aldolase B was shown in patients without liver metastasis.

Topics: Chloramines; Fructose-Bisphosphate Aldolase; Humans; Isoenzymes; Liver Diseases; Radioimmunoassay; Time Factors; Tissue Distribution; Tosyl Compounds; Urea

Topics: Bleomycin; Chloramines; Imidazoles; Iodine Radioisotopes; Isotope Labeling; Peplomycin; Radioimmunoassay; Tosyl Compounds; Urea

We have studied the behaviour of 125I-labelled alpha-MSH under different experimental conditions. Until now, the chloramine T method had been used by most investigators with variable results. We have tested three other labelling techniques based on 125I mild oxidation: (1) an enzymatic method with lactoperoxidase, (2) a sparingly soluble chloramine method (T.D.G.U.) and (3) modified chloramine T procedure, 'the iodine volatilization method'. Labelled hormone obtained after each kind of iodination was assayed for immunoreactivity. In addition, time course degradation was measured by classical RIA incubation procedures. Charcoal-dextran was used to separate bound and free antigen. We have found chloramine T-iodinated alpha-MSH to be significantly more damaged than preparations obtained by other methods and to be less stable when stored at -18 degrees C. No differences were found between the differently labelled 125I-labelled alpha-MSH fresh preparations in binding to surface receptors of human melanoma cell lines in culture.

Topics: Animals; Cattle; Chloramines; Chromatography, Gel; Humans; Immune Sera; Immunologic Techniques; Iodine Radioisotopes; Isotope Labeling; Lactoperoxidase; Melanocyte-Stimulating Hormones; Melanoma; Rabbits; Tosyl Compounds; Urea; Volatilization

Topics: Chloramines; Chromatography, Gel; Escherichia coli; Iodine Radioisotopes; Isotope Labeling; Muramidase; Oxidation-Reduction; Protein Binding; Ribosomes; Staphylococcal Protein A; Tosyl Compounds; Urea

Different states of Dipetalonema viteae (males, females, microfilariae, and 3rd-stage larvae) have been iodinated in vitro under physiological conditions by chloroglycoluril, lactoperoxidase or chloramine T. The concentrations of the catalysts were correlated with the viability of the worms. Localization of the label with the different iodination methods had been visualized by electro microscopical autoradiography. Chloroglycoluril-mediated iodination is predominantly localized on the filarial cuticle. Lactoperoxidase-catalysed iodination is less specific and chloramine T catalyses iodination in a gradient decreasing from the cuticle to inner structures. It is necessary to visualize the labelling by electron microscopical autoradiography prior to biochemical and immunological experiments to avoid the extraction of structures iodinated by leakage of the catalyst into sub-cuticular regions.

Topics: Animals; Antigens, Surface; Autoradiography; Chloramines; Dipetalonema; Female; Iodine Radioisotopes; Lactoperoxidase; Male; Microscopy, Electron; Tosyl Compounds; Urea

Topics: alpha-Fetoproteins; Chloramines; Chromatography, Affinity; Costs and Cost Analysis; Humans; Indicators and Reagents; Iodine Radioisotopes; Radioimmunoassay; Tosyl Compounds; Urea

A comparison of labelling compounds with chloramine-T and with 1, 3, 4, 6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodogen) has been carried out. For human transferrin, human calcitonin, 1-84 bovine parathyrin, fibrinopeptide-A, human thyrotropin and F-CB3, a cyanogen bromide cleavage peptide of human fibrinogen, the quality of tracer produced by the Iodogen method was better. For rat lutropin, human growth hormone and human prolactin, labelling with Iodogen produced a tracer of unsatisfactory quality. For a further 13 peptides, the results from both methods were comparable. Optimal reaction times using Iodogen were of the magnitude of two to three times longer than when using chloramine-T. Reduction of the volume of radioactive waste by up to 90% could be achieved when the Iodogen method was coupled with a short cation-exchange column to separate unreacted iodide from the labelled compound. Data is presented on the quality of tracer, expressed in terms of elution profiles and radioimmunoassay standard curves. A novel "combi-method" of labelling proteins without tyrosine or histidine moieties is presented where N-succinimidyl-3-(4-hydroxyphenyl)-propionate is labelled at pH 7.5 using Iodogen to give "Bolton-Hunter" reagent, which is then transferred to a vessel containing the peptide to be labelled at ph 8.6.

Topics: Animals; Blood Proteins; Chloramines; Humans; Imidazoles; Indicators and Reagents; Iodine Radioisotopes; Isotope Labeling; Radioimmunoassay; Rats; Tosyl Compounds; Urea