BINDING OF ZINC TO INSULIN AND SOME INSULIN DERIVATIVES STUDIED BY PAPER ELECTROPHORESIS

ABSTRACT Paper electrophoretic fractionation in barbiturate (barbital) buffer, pH 9, of iodine-substituted insulin 0–10.8 I/mole showed that substitution with 4–6 I/mole influences the binding of zinc to a demonstrable extent. The effect appears to be due to substitution in the imidazole groups of the histidine residues. Substitution with iodine in the tyrosine residues seems to be without significance, at least at the lower degrees of iodination. The importance of the histidine residues for the binding of zinc is shown by selective destruction of the imidazole groups by photo-oxidation, sensitized by methylene blue. Carbamylation of the N-terminal α-amino groups in the A- and B-chains with KOCN only slightly influences the ability to bind zinc while carbamylation with fluorescein isothiocyanate in the N-terminal of the B-chain brings about a more pronounced reduction in the zinc binding capacity.

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METHODOLOGICAL STUDY OF THE RADIOIMMUNOASSAY OF HUMAN THYROTROPHIN

Acta Endocrinologica ◽

10.1530/acta.0.0710024 ◽

1972 ◽

Vol 71 (1) ◽

pp. 24-36 ◽

Cited By ~ 24

Author(s):

Ariel Gordin ◽

Pirkko Saarinen

Keyword(s):

Healthy Subjects ◽

Binding Capacity ◽

Paper Electrophoresis ◽

Storage Conditions ◽

Methodological Study ◽

Double Antibody ◽

The Mean ◽

Specific Activities ◽

Initial Binding ◽

In Pregnancy

ABSTRACT An account is given of a methodological study of the double-antibody radioimmunoassay of human TSH, using highly purified labelled human TSH as tracer. It was shown that conventional paper electrophoresis was not adequate for studying the purity of labelled human TSH. When polyvinylchloride (Pevikon®) electrophoresis was used, four subfractions could still be separated, even though, on paper electrophoresis, the material seemed to be homogeneous. Only two of the four Pevikon fractions were immunoreactive. Purification of labelled human TSH by Pevikon electrophoresis also improved the sensitivity of the assay. Specific activities of about 100 mCi/mg gave the highest initial binding capacity, produced least damage to the labelled hormone and showed the best stability of the tracer without influencing the sensitivity of the method. In different storage conditions, labelled human TSH was found to be most stable at −20°C and diluted 1/100. Only in pregnancy did the addition of HCG seem necessary. The mean TSH value in healthy subjects was 3.6 ± 1.4 μU/ml (mean±sd) with a range from 1.6 μU/ml to 8.8 μU/ml.

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Reaction of Fluorescein Isothiocyanate with Thiol and Amino Groups of Sarcoplasmic ATPase

Zeitschrift für Naturforschung C ◽

10.1515/znc-1985-11-1220 ◽

1985 ◽

Vol 40 (11-12) ◽

pp. 863-875 ◽

Cited By ~ 22

Author(s):

Gertrude Swoboda ◽

Wilhelm Hasselbach

Keyword(s):

Absorption Maximum ◽

Sodium Dodecylsulfate ◽

Specific Effect ◽

Fluorescein Isothiocyanate ◽

Thiol Groups ◽

Amino Groups ◽

Model Compounds ◽

Intramolecular Transfer ◽

Dodecyl Ether ◽

Lysyl Residue

Abstract Several model compounds containing thiol and/or amino groups (mercaptoethanol, glutathione, cysteine, ethanolamine, glycine) were studied with respect to their reactivity towards fluorescein isothiocyanate (followed spectrophotometrically at 504 and 412 nm), stability of product and long wave absorption maximum of the fluorescein residue attached. Thiol groups reacted by far more readily than amino groups. A specific effect was observed with cysteine, indicating an intramolecular transfer of the fluorescein residue from SH to NH2.With sarcoplasmic vesicles both types of reactions were observed. The ratio of products, which can be distinguished by their different stabilities and absorption spectra, depended on the absence or presence of detergents. While with native vesicles the NH2 reaction predominated, with vesicles solubilized with sodium dodecylsulfate, octaethyleneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine the SH reaction became prevailing. Already 0.35 mg sodium dodecylsulfate per mg protein were sufficient to give rise to dithiourethane formation exclusively. Excess fluorescein isothiocyanate reacted with several thiol groups of dodecylsulfate-solubilized vesicles. In the presence of ATP binding of fluorescein isothiocyanate to native vesicles was significantly reduced.Total blockage of the vesicular SH groups with N-ethyl-maleimide led to preparations that reacted with fluorescein isothiocyanate much more slowly, compared to native vesicles. Octaethy leneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine in the assay accelerated the thioureide formation from N-ethylmaleimide modified vesicles, whereas sodium dodecylsulfate prevented it almost completely.Our results support the suggestion that one or several thiol groups in vicinity of the highly reactive lysyl residue might play a role in the fast specific reaction, which is only observed with intact native vesicles.

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Erratum

Clinical Chemistry ◽

10.1093/clinchem/5.6.621 ◽

1959 ◽

Vol 5 (6) ◽

pp. 621-621

Keyword(s):

Medical School ◽

Paper Electrophoresis ◽

Buffer System ◽

Journal Issue ◽

Sample Application ◽

Barbital Buffer

Abstract A Discontinuous Buffer System for Paper Electrophoresis of Human Hemoglobins C. A. J. Goldberg The figure for this paper, published in the last journal issue, has been reproduced below by another engraving process in order to more fully illustrate the text of the article. The Editor Fig. 1. Paper electrophoresis of human hemoglobins in a continuous barbital buffer system (as shown in patterns 1 and 3) and in a discontinuous buffer system of Tris-EDTA-borate (TEB) and barbital (see text) as shown in patterns 2 and 4 to 8. The migration of the hemoglobins is anodic in both systems. The site of sample application is at the top of the patterns, and is visible in patterns 1 and 3. The sample of hemoglobins H trait was received through the courtesy of Dr. D. A. Rigas, University of Oregon Medical School, Portland, Ore.

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Chemical Modifications of Escherichia coli Ribosomal Proteins S4, S7, and S8 in Regard to Their Capacity for Binding 16 S RNA

Canadian Journal of Biochemistry ◽

10.1139/o74-145 ◽

1974 ◽

Vol 52 (11) ◽

pp. 1038-1043 ◽

Cited By ~ 4

Author(s):

Gérald Lemieux

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Binding Capacity ◽

Cysteine Residue ◽

Amino Groups ◽

Chemical Modifications ◽

Specific Chemical ◽

Rna Interaction ◽

Rna Complex ◽

Rna Complexes

Mild specific chemical modifications were used to test the possible role of cysteines, tyrosines, and amino groups of ribosomal proteins S4, S7, and S8 for binding to 16 S RNA (Escherichia coli). The single cysteine residue present in proteins S4 and S8 is not directly involved in the binding process with RNA. The protein S7 does not contain cysteine. Total nitration of tyrosines in S4, S7, and S8 abolishes the binding capacity of these proteins. However, when the modification is done with the preformed protein–RNA complexes, some protection occurs. Two amino groups could be reacted in free S8 as well as in an S8–RNA complex. This modification does not influence protein–RNA interaction. Proteins S4, S7, and S8 in Regard to Their Capacity for Binding 16 S RNA. Can. J. Biochem. 52, modified. A new method for the stoichiometric determination of single protein–RNA complexes is presented.

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Involvement of the Zinc-Binding Capacity of Sendai Virus V Protein in Viral Pathogenesis

Journal of Virology ◽

10.1128/jvi.74.17.7834-7841.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 7834-7841 ◽

Cited By ~ 43

Author(s):

Cheng Huang ◽

Katsuhiro Kiyotani ◽

Yutaka Fujii ◽

Noriko Fukuhara ◽

Atsushi Kato ◽

...

Keyword(s):

Fusion Proteins ◽

Sendai Virus ◽

Binding Capacity ◽

Viral Pathogenesis ◽

Growth Patterns ◽

Zinc Binding ◽

Wild Type ◽

V Protein ◽

Rich Domain ◽

Cysteine Rich Domain

ABSTRACT The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C341S and V-C365R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV VΔC, which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.

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QS389. Disturbances in Free Zinc Concentration and Zinc Binding Capacity of the Plasma in Hemorrhagic Shock

Journal of Surgical Research ◽

10.1016/j.jss.2008.11.699 ◽

2009 ◽

Vol 151 (2) ◽

pp. 300

Author(s):

J.E. Kohler ◽

J. Mathew ◽

A.L. Blass ◽

D.I. Soybel ◽

E. Kelly

Keyword(s):

Hemorrhagic Shock ◽

Zinc Concentration ◽

Binding Capacity ◽

Zinc Binding ◽

Free Zinc

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Kinetic properties of the α-lytic protease of Sorangium sp., a bacterial homologue of the pancreatopeptidases

Canadian Journal of Biochemistry ◽

10.1139/o69-047 ◽

1969 ◽

Vol 47 (3) ◽

pp. 305-316 ◽

Cited By ~ 26

Author(s):

H. Kaplan ◽

D. R. Whitaker

Keyword(s):

Methyl Ester ◽

Catalytic Properties ◽

Substrate Binding ◽

Kinetic Properties ◽

Histidine Residue ◽

Amino Groups ◽

Binding Properties ◽

Histidine Residues ◽

Support Reaction ◽

Hydrolysis Of

The kinetics under consideration are those of a bacterial serine protease with the same "active serine" sequence as chymotrypsin, trypsin, and elastase, and with a single histidine residue in a sequence which closely matches the sequences around histidine-57 of chymotrypsin and the analogous histidine residues of trypsin and elastase. In agreement with previous evidence of an elastase-like specificity, esters of N-substituted, neutral, aliphatic L-amino acids proved to be good to excellent substrates for the α-enzyme; esters of arginine, tyrosine, and tryptophan were not hydrolyzed. The enzyme has a much higher activity than the pancreatopeptidases towards p-nitrophenyl acetate and p-nitrophenyl trimethyl acetate; the catalytic rate coefficient kc for the latter substrate is about fivefold greater than that of elastase.The catalytic properties match those of the pancreatopeptidases in the following respects. As demonstrated with N-acetyl-L-valine methyl ester as substrate, kc is dependent on an ionization with a pKa of 6.7 in water and 7.3 in H22O; Δ log (kc/Km)/ΔpH for this ionization is equal to 1.0; kc is reduced 50% when H2O is replaced by H22O. These findings are consistent with a requirement for a single unprotonated histidine residue and general basic catalysis by that residue. The burst of p-nitrophenol in hydrolyses of p-nitrophenyl trimethyl acetate is proportional to [E]0; the magnitude of the proportionality factor and the rate of attainment of a steady state are consistent with the condition [Formula: see text], as in chymotrypsin kinetics. Thus the purely catalytic properties of the α-enzyme match those of chymotrypsin very closely. These findings do not support reaction mechanisms which require two catalytically functional histidine residues for such catalysis. The substrate-binding properties of the α-enzyme differ from those of chymotrypsin in that substrate binding does not depend on ionization of an N-terminal α-amino group; Km for the hydrolysis of N-acetyl-L-valine methyl ester is constant from pH 5 to pH 10 and enzymatic activity is unaffected by acetylation of the enzyme's α- and ε-amino groups. Ks for the hydrolysis of p-nitrophenyl trimethyl acetate is appreciably greater than the Ks of elastase for this substrate.The chloromethyl ketones of glycine and valine did not inhibit the enzyme or alkylate its histidine residue.

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SPECIFIC BINDING OF THYROID-STIMULATING HORMONE BY HUMAN SERUM GLOBULINS

Journal of Endocrinology ◽

10.1677/joe.0.0880339 ◽

1981 ◽

Vol 88 (3) ◽

pp. 339-349 ◽

Cited By ~ 22

Author(s):

J. BÍRÓ

Keyword(s):

Binding Sites ◽

Binding Capacity ◽

Specific Binding ◽

Negative Control Group ◽

Paper Electrophoresis ◽

Negative Control ◽

Thyroid Stimulating Hormone ◽

Human Thyroid ◽

Control Group ◽

Binding Characteristics

Globulin preparations (41) from patients with Graves's disease (positive to thyroid stimulating immunoglobulins; TSI) and 12 from healthy persons (TSI-negative) were tested for their specific thyrotrophin (TSH)-binding properties. Globulins from both groups possessed binding sites for 131I-labelled TSH. The mean dissociation constant (Kd) was 6·8 pmol/l per mg globulin and the maximum specific binding (Bmax) was 3·0 pmol/mg globulin per 1 for the TSI-negative control group. Twenty-four (58·5%) globulin preparations from the TSI-positive group had similar TSH-binding characteristics with mean Kd of 7·2 pmol/l per mg globulin and Bmax of 3·6 pmol/mg globulin per 1 (A-type binding) but the remaining 17 (41·5%) bound TSH in a different fashion with Kd of 71·5 pmol/l per mg globulin and Bmax of 13·6 pmol/mg globulin per 1 (B-type binding). Both types of specific TSH binding reached the maximal level within 1 h of incubation and had an optimum pH of 7–8. There was a linear correlation between the amount of bound TSH and the globulin content of the samples. Both types of binding were reversible by the addition of an excess of TSH and gonadotrophins, ACTH, prolactin and insulin competed with TSH for the binding sites only when in relatively high concentrations. The binding sites were associated with macromolecules; they emerged with the void volume after chromatography on Sephadex G-200 and migrated with immunoglobulin G (IgG) on paper electrophoresis. The binding capacity of the globulin preparations could be decreased by preincubation with antiserum to human IgG or with human thyroid membranes.

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Kinetics of the Reaction of Nitrous Acid with Model Compounds and Proteins, and the Conformational State of N-terminal Groups in the Chymotrypsin Family

Canadian Journal of Biochemistry ◽

10.1139/o72-174 ◽

1972 ◽

Vol 50 (12) ◽

pp. 1282-1296 ◽

Cited By ~ 26

Author(s):

A. Kurosky ◽

T. Hofmann

Keyword(s):

Nitrous Acid ◽

Conformational State ◽

Serine Proteinases ◽

Amino Groups ◽

Model Compounds ◽

Third Order ◽

Tyrosine Residues ◽

Reactive Groups ◽

Primary Amino ◽

Kinetics Of

The kinetics of the reaction of nitrous acid at 4° and pH 4.0 with various amino acids, peptides, and proteins were studied. The reaction with isoleucine methyl ester was found to have a linear dependence on the square of the HONO concentration showing that N2O3 was the reactive species. Third order nitrosation rate constants of primary amino groups showed a correlation with their pK values. They were calculated for the concentration of the unprotonated species to give intrinsic reactivities. The rate of nitrosation of acetyltryptophan to give N-nitrosoacetyltryptophan was found to be a linear function of the nitrous acid concentration. This nitrosation therefore follows a different mechanism. The reaction of nitrous acid with tyrosine residues was examined by spectrophotometry. The reaction was negligible compared to that of other groups. Acetylhistidine and imidazole did not react. Reactivities for α-amino groups, ε-amino groups, and other residues in proteins were compared. The conformational state of the N-terminal residues in serine proteinases, as revealed from their reactivities, is discussed in detail. It is concluded that nitrous acid reacts preferentially with "surface" residues and is a useful tool for exploring conformational states of reactive groups in proteins, especially α-amino groups and indole rings.

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