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PubMed1983

Synthesis of a 125I-radiolabeled penicillin for penicillin-binding proteins studies.

Radioiodination of penicillin X (p-hydroxybenzylpenicillin) with 125INa, using the chloramine-T method, is simple and almost quantitative. The product thus obtained can be used without further purification for the penicillin-binding proteins (PBPs) assay. The chemical structure of 125I-penicillin X is very close to that of penicillin G, and the affinity of the two compounds for the PBPs are very similar. 125I-penicillin X can then advantageously replace [14C]penicillin G in these experiments, as its specific radioactivity is 2000 Ci/mol, in our preparations, instead of about 50 Ci/mol; thus, the experimental procedure is simplified and the autoradiography exposure time is reduced to 12-48 h.

Autoradiography↗
PubMed1983

Labeling of platelet surface proteins with 125I by the iodogen method.

A procedure for the 125I-iodination of platelet suspensions is described. The procedure utilizes Iodogen, a solid-phase oxidizing agent similar to chloramine-T. Platelets were labeled under a variety of conditions, including in the presence of 0.1% albumin, and showed between 7 and 28% incorporation of 125I. Best labeling results were obtained at low platelet concentrations (3-5 x 10(8) platelets/ml), short reaction times (15 min), and with 2-ml glass vials coated with 100 micrograms of Iodogen. Analysis of the labeled platelet proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed that the same major protein bands were labeled by this procedure as were labeled by the lactoperoxidase procedure. At low platelet concentrations, the Iodogen procedure gives twice the amount of iodine incorporation.

Blood Platelets↗
PubMed1986

A versatile transition metal salt reaction for a wide range of common biochemical reagents: an instantaneous and quantifiable color test.

A rapid and sensitive spot test amenable to visual or spectrophotometric quantitation has been developed for a wide variety of biochemical reagents by utilizing the transition metal salt cupric chloride and its large number of related colored compounds. This assay is potentially a widely applicable multipurpose test for rapidly detecting the presence of unknown substances. Combination of the test sample with the working reagent results in the immediate formation of a distinctive colored product that may be precipitable. Some compounds require the further addition of sodium hydroxide in order to generate the distinctively colored product. Distinctive reactions occur with the following reagents, and their limit of visual detection is indicated in parentheses: ammonium bicarbonate (12.5 mM), ammonium acetate (25 mM), ammonium hydroxide (0.1%), ammonium sulfate (2%), ammonium persulfate (0.02 mM), L-(+)-cysteine (0.07 mM), dithiothreitol (DTT) (1.25 mM), EDTA (0.6 mM), ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (5 mM), D-glucose (6 mM), glycerol (0.3%), imidazol (12.5 mM), DL-methionine (100 mM), mercaptoethanol (0.05%), sodium azide (19 mM, 0.1%), sodium dithionite (0.25%), sodium metabisulfite (25 mM), sodium nitrite (6.2 mM), sodium periodate (3.1 mM), sodium sulfite (12.5 mM), sodium thiosulfite (12.5 mM), sucrose (6 mM), and N,N,N',N'-tetramethylethylenediamine (0.05%). A distinctive exothermic reaction occurs with hydrogen peroxide, but without color change. Compounds reacting insignificantly include 50 mM Tris buffer, urea, N,N'-methylene bisacrylamide, sodium dodecyl sulfate, isopropyl alcohol, sodium fluoride, trichloroacetic acid, phenol, mannose, K2HPO4, guanidine HCl, chloramine-T, magnesium chloride, and boric acid, where the solids were tested at approximately 10 mg/ml. Spectrophotometric standard curves were developed for DTT and sodium azide utilizing the clear supernatants resulting from these reactions. Combinations of at least four reagents could be discriminated, as demonstrated with mixtures of glucose, sodium azide, EDTA, and DTT. In addition ammonium sulfate could be detected to a limit of 4% in the presence of protein, DTT, and EDTA in a 50 mM Tris buffer. Spot tests were developed which utilized reagent-impregnated filter paper and gave distinctive colored products on addition of 5 microliter of test sample.

Centrifugation↗
PubMed1987

Improved synthesis and iodination of a cleavable photoactivated probe.

J. B. Denny and G. Blobel (1984, Proc, Natl. Acad. Sci. USA 81, 5286-5290) have described the synthesis of a water-soluble novel heterobifunctional 125I-labeled photocrosslinking reagent, N-[4-(p-azido-m-[125I]iodophenylazo)benzoyl]-3-aminopropionyl-N'- oxysulfosuccinimide, which contains a cleavable internal azo bond. We report several modifications of their synthesis which greatly increase the yield of a synthetic intermediate, N-[4-(p-aminophenylazo)benzoyl]-3-aminopropionic acid (compound VII). Evidence is presented that direct iodination of compound VII with chloramine-T produces low yields of 125I-labeled compound VII in a reaction which is difficult to control. Alternatively, the yield of 125I-labeled reagent can be greatly improved if the iodination is performed following the derivatization of N-[4-(p-axidophenylazo)benzoyl]-3-aminopropionyl-N'-oxysulfosuc cinimide (compound IX) to a ligand such as dextran. We have demonstrated the transfer of 125I label from the reagent when derivatized to amino-dextran to proteins in solution after photolysis and cleavage of this 125I-labeled reagent.

Chemical Phenomena↗
PubMed1990

Site specific radioiodination of recombinant hirudin.

Recombinant hirudin variant rHV2-Lys47 was radioiodinated using the chloramine-T method. Depending on the reaction pH, the two tyrosine residues, Tyr3 and Tyr63, responded differently to iodination but without change in total iodination yield. Of the incorporated -125 iodine 80% was located on Tyr3 at pH 7.4, but 65% was found on Tyr63 at pH 4. These distinct iodination patterns suggest the existence of a pH-dependent multimerization and/or important conformational changes in the tertiary structure with pH. Each radiotracer was purified to high specific activity by simple low-pressure chromatography including gel filtration and reverse-phase separation, both on short cartridges. The method was validated by reverse-phase and anion-exchange HPLC with on-line radioactivity detection. The iodination sites were characterized following carboxypeptidase Y cleavage coupled with radio-HPLC.

Amino Acid Sequence↗
PubMed1986

Immunological and chemical properties of mouse alpha 1-protease inhibitors.

We previously described the isolation and purification of two similar alpha 1-protease inhibitors from mouse plasma termed alpha 1-PI(E) and alpha 1-PI(T) because of their respective affinities for elastase and trypsin. Some of the biochemical and immunological properties of these proteins are reported. Both are acidic glycoproteins with pI's of 4.1-4.2. The plasma half-life of each inhibitor, determined after administration of the 125I-protein, is approximately 4 h both in normal mice and in mice after induction of the acute phase reaction. The two proteins have almost identical amino acid compositions and similar CNBr peptide maps. Tryptic maps, however, are considerably different. Reverse-phase chromatography separated alpha 1-PI(E) into three distinct isoforms, each eluting with approximately 60% acetonitrile. Under these conditions alpha 1-PI(T) shows a single peak, clearly different from those of alpha 1-PI(E). The three alpha 1-PI(E) isoforms have the same molecular weights on sodium dodecyl sulfate-gel electrophoresis and the same tripeptide sequence at their N-terminus, and appear to be immunologically identical. Polyclonal, monospecific antibodies to each native inhibitor, prepared in rabbits, showed no cross-reactivity when tested by functional assay or crossed immunoelectrophoresis. Interestingly, each antibody recognized epitopes on the C-terminal portion of its respective antigen. These studies confirm that alpha 1-PI(E) and alpha 1-PI(T), although highly similar, are products of different genes. Like human alpha 1-PI, the two mouse inhibitors are partially inactivated by mild oxidation with chloramine-T, losing all elastase inhibitor and lesser amounts of antichymotryptic and antitryptic activity. However, unlike the human protein, neither alpha 1-PI(E) nor alpha 1-PI(T) was found to have a methionine residue at its P1 site.

Amino Acid Sequence↗
PubMed1986

Role of ubiquitin conformations in the specificity of protein degradation: iodinated derivatives with altered conformations and activities.

Three iodinated derivatives of ubiquitin have been synthesized and these derivatives have been characterized in the ubiquitin-dependent protein degradation system. With chloramine-T as the oxidant, a derivative containing monoiodotyrosine is formed in the presence of 1 M KI and a derivative containing diiodotyrosine is produced in the presence of 1 mM KI. These derivatives exhibit phenolate ionizations at pH 9.2 and 7.9 with absorbance maxima at 305 and 314 nm, respectively. In addition to modification of the tyrosine residue, these conditions lead to the oxidation of the single methionine residue and iodination of the single histidine residue [M.J. Cox, R. Shapira, and K.D. Wilkinson (1986) Anal. Biochem. 154, 345-352]. Iodination of ubiquitin under these conditions renders the protein sensitive to hydrolysis by trypsin and results in an enhanced susceptibility to alcohol-induced helix formation. When the derivatives are tested in the ATP: pyrophosphate exchange reaction catalyzed by the ubiquitin adenylating enzyme, they are found to exhibit activity comparable to the native protein. When these derivatives are tested for the ability to act as a cofactor in the ubiquitin-dependent protein degradation system, they are both found to support a rate of protein degradation that is twice that of native ubiquitin. At high concentrations of derivatives, the rate of protein degradation is inhibited, while the steady state level of conjugates increases. Thus, the free derivatives inhibit the protease portion of the reaction, but are fully active in the activation and conjugation portions of the reaction. With iodine as the modification reagent, monoiodination of tyrosine is the predominant reaction. This derivative exhibits activity similar to native ubiquitin. Thus, it appears that modification of the histidine residue is responsible for the increased activity of the more highly iodinated derivatives. The enzymes of the system must recognize different portions of the ubiquitin structure, or different conformations of ubiquitin that are affected by the iodination of the histidine residue. These results suggest a conformational change of the ubiquitin molecule may be important in determining the rate and specificity of proteolysis.

Circular Dichroism↗
PubMed1990

Purification and characterization of bovine cerebral cortex A1 adenosine receptor.

A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine greater than (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA greater than NECA greater than S-PIA. The A1AR was solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy]-phenyl]-1,3- dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand [( 3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax greater than 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA greater than S-PIA greater than NECA. The overall yield from soluble preparation was 7%. The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to alpha-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes.

Animals↗
PubMed1976

125I derivatives of prostaglandins. A novel approach in prostaglandin analysis by radioimmunoassay.

We report the production of radioactive iodinated (125 I) derivatives of prostaglandins E1, E2, F2alpha and their use in radioimmunological assays. Histamine or tyramine was coupled to the prostaglandins carboxyl group and the iodination was accomplished using the chloramine T method. The high specific radioactivity of these tracers and the resolution of the purification procedure allowed the detection of 0.5 pg of prostaglandins. A comparison with tritiated prostaglandin was made and showed a 10-fold gain in sensitivity. Furthermore in the case of the prostaglandin E1 system using 125I-labelled histamine or tyramine as tracer the cross reaction curves obtained were different from those obtained with [3H]prostaglandin E1; we suggest that the blocking of the carboxyl group alters the prostaglandin E1 structure, modifying its immunoreactivity.

Amniotic Fluid↗
PubMed1985

Radioiodination of naturally occurring phospholipids.

A simple and rapid nonenzymatic method for radioiodination of phospholipids is described. It involves oxidation of Na125I with TlCl3 (or chloramine-T) in an aqueous medium, with subsequent exposure of the phospholipids, dissolved in chloroform/methanol, to the action of the oxidizing mixture. Purification of the radiolabelled phospholipids was effected by washing with sodium thiosulphate followed by thin-layer chromatography on silica gel. Specific radioactivity of 125I-labelled phosphatidylcholine was estimated to be about 10 muCi/mg phospholipid. The method is designed for radioiodination of various naturally occurring phospholipids.

Chromatography, Thin Layer↗
PubMed1976

Radioimmunoassay of apolipoprotein A-I of rat serum.

A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.

Animals↗
PubMed1977

Radioimmunoassay of arginine-rich apolipoprotein of rat serum.

A double-antibody radioimmunoassay was developed for quantification of rat arginine-rich apolipoprotein in sodium decyl sulfate. Arginine-rich protein, labeled with 125I by the chloramine-T method, had the same chromatographic characteristics on Sephadex G-200 as unlabeled arginine-rich protein and up to 70% of 125I-labeled arginine-rich protein was precipitated by antisera to arginine-rich protein in rabbits. The assay is sensitive at the level of 1-10 ng and has intraassay and interassay coefficients of variation of 5.4 and 6.8%, respectively. The specificity of the assay was established by competitive displacement of 125I-labeled arginine-rich protein from its antiserum by arginine-rich protein and lipoproteins containing this protein, but not by rat albumin or other purified apolipoproteins. Immunoreactivity of rat serum and lipoproteins was complete as demonstrated by comparison with their delipidated form. The accuracy of the immunoassay was further substantiated by comparison with the amount of arginine-rich protein in chromatographic fractions of total apoprotein of very low and high density lipoproteins, and by recovery experiments in ultracentrifugally separated fractions of serum. In contrast to an immunoassay reported previously for rat apo A-I, sodium decyl sulfate was not required for complete immunoreactivity of serum and lipoproteins. The inclusion of sodium decyl sulfate (9 mM final concentration) was necessary, however, for stability of labeled and unlabeled preparations of arginine-rich protein. Content (weight %, means values +/- S.D.), of immunoassayable arginine-rich protein in isolated lipoproteins was 15 +/- 1.5% in very density lipoproteins; 6.8% in low density lipoproteins (1.02 less than d less than 1.04 g/m); 7.1 +/- 0.3% in high density lipoproteins; and 4.8 +/- 0.5% in lymph chylomicrons. Concentration in whole serum was 18.1 +/- 1.4 and 20.4 +/- 2.3 mg/dl for male and female rats, respectively. Only about 55% of arginine-rich protein was recovered in the major lipoprotein classes and about 40% was in "lipoprotein-free" serum (d greater than 1.25 g/ml). Among the lipoproteins, the high density lipoprotein fraction contained twice the amount of arginine-rich protein recovered in very low or low density lipoproteins (26.6 vs. 13.5 and 13.4%, respectively). The significance of the large amount of arginine-rich protein in the 1.25 g/ml infranatant fraction is not apparent. Although repetitive centrifugation did not alter the amount recovered in this fraction, the possibility of an artifact induced by centrifugation and high salt concentration cannot be excluded.

Amino Acids↗
PubMed1979

Comparative study between prokaryotes and eukaryotes by chemical iodination of ribosomal proteins.

Escherichia coli and Saccharomyces cerevisiae ribosomal proteins were chemically iodinated with 125I by chloramine T under conditions in which the proteins were denatured. The labelled proteins were subsequently separated by two-dimensional gel electrophoresis with an excess of untreated ribosomal proteins from the same species. The iodination did not change the electrophoretic mobility of the proteins as shown by the pattern of spots in the stained gel slabs and their autoradiography. The 125I radioactivity incorporated in the proteins was estimated by cutting out the gel spots from the two-dimensional electrophoresis gel slabs. The highest content of 125I was found in the ribosomal proteins L2, L11, L13, L20/S12, S4 and S9 from E. coli, and L2/L3, L4/L6/S7, L5, L19/L20, L22/S17, L29/S27, L35/L37 and S14/S15 from S. cerevisiae. Comparisons between the electrophoretic patterns of E. coli and S. cerevisiae ribosomal proteins were carried out by coelectrophoresis of labelled and unlabelled proteins from both species. E. coli ribosomal proteins L5, L11, L20, S2, S3 and S15/S16 were found to overlap with L15, L11/L16, L36/L37, S3, S10 and S33 from S. cerevisiae, respectively. Similar coelectrophoresis of E. coli 125I-labelled proteins with unlabelled rat liver and wheat germ ribosomal proteins showed the former to overlap with proteins L1, L11, L14, L16, L19, L20 and the latter with L2, L5, L6, L15, L17 from E. coli.

Animals↗
PubMed1983

Effects of common radioiodination procedures on the binding of glycoproteins to immobilized lectins.

Representative glycoproteins including fetuin, protein A, ovalbumin, alpha 1 acid glycoprotein, and the major glycoprotein of equine infectious anemia virus were labelled with 125I by the chloramine-T or Bolton-Hunter procedure and their binding to immobilized Con A or lentil lectin compared to untreated samples of each glycoprotein. Glycoprotein modification was no greater than one substituted residue per protein molecule. Yet the radioiodinated glycoproteins typically displayed only 0-50% of the lectin binding observed with untreated samples. These results indicate that lectin glycoprotein binding can be markedly altered by minor modifications in protein structure.

Chromatography, Affinity↗
PubMed1985

Role of one tryptophan residue in the lethal activity of Clostridium perfringens epsilon toxin.

The lethal activity of Clostridium perfringens epsilon toxin was inactivated by N-bromosuccinimide and N-chlorosuccinimide. Amino acid analysis of N-bromosuccinimide-treated prototoxin indicated that the one tryptophan residue present in the protein was abolished, and methionine and tyrosine reduced markedly. N-chloro-succinimide-treated prototoxin lost completely both tryptophan and methionine residues. The toxin was not inactivated by chloramine T, but all of methionine residues present in the protein was found to be oxidized by the agent. The data suggest that the one tryptophan residue present in the toxin is important for the lethal activity.

Amino Acids↗
PubMed1985

Fast and efficient method for detection and estimation of proteins.

A quick, simple, inexpensive and sensitive method is described to stain and quantitate proteins on nitrocellulose papers. The proteins may be spotted or transferred from polyacrylamide gels by Western blotting. The procedure involves non-radioactive iodination of the polypeptides by chloramine T and potassium iodide followed by detection of bound iodine with starch. The method is more sensitive and much quicker than Coomassie brilliant blue staining and may be used for quantitation or detection of proteins in unknown samples. Another major advantage of this procedure is that ionic or nonionic detergents, although at higher concentrations causing the sample to disperse more broadly in the membranes, do not affect the staining procedure. Further, this method may be used for detection of proteins bound to papers that have high affinity for proteins such as the Zeta probe membranes.

Candida albicans↗
PubMed1987

High molecular weight Alzheimer's disease amyloid peptide immunoreactivity in human serum and CSF is an immunoglobulin G.

A radioimmunoassay (RIA) was developed to detect the 4200 Dalton amyloid (A4) peptide or it's precursor (A4P) in human serum or cerebrospinal fluid (CSF). A synthetic peptide containing the first 28 amino acids of the 43 amino acid A4 peptide was covalently coupled to bovine thyroglobulin and a polyclonal antiserum in rabbits was prepared. This antiserum was specific for vascular amyloid and neuritic plaques in Alzheimer's disease brain as detected by immunoperoxidase. The synthetic peptide, which has a tyrosine at residue 10, was iodinated with chloramine T and [125I]iodine and was purified to homogeneity by C4 reverse phase high performance liquid chromatography (HPLC). Extraction of human serum over a C18 Sep Pak cartridge indicated immunoreactive A4 peptide was not detectable in human serum. Conversely, high molecular weight A4 peptide immunoreactivity was detectable in human serum, at a concentration of 8.9 +/- 1.2 pmol-eq./ml, and in human CSF, at a concentration of 0.25 +/- 0.01 pmol-eq./ml, giving a CSF/serum ratio of 3.2%. The immunoreactivity in human serum was nearly completely removed by affinity deletion of serum immunoglobulin G (IgG), but not by affinity removal of IgA or IgM. Serum immunoreactivity was decreased 90% in hypogammaglobulinemia, and was increased 83% in human cord serum. There was no statistical difference in serum A4 immunoreactivity in Alzheimer's serum or CSF. Serum immunoreactivity in Down's syndrome was increased 50%. These studies indicate the high molecular weight A4P immunoreactivity in human serum or CSF is an IgG. Whether the A4 precursor in Alzheimer's disease is, in fact, an IgG, or whether there is an antibody in human serum and CSF that cross reacts with the A4 precursor cannot be determined until the serum immunoreactivity is purified and structurally characterized.

Alzheimer Disease↗
PubMed1992

Synthesis and biochemical properties of an 125I-labelled ryanodine derivative.

The synthesis of a novel radioiodinated ryanodine-O10eq-N-acylamino acylate along with biological data are reported. The affinity of the iodinated product, 7, was comparable to ryanodine, 7.97 nM and 6.47 nM, respectively. Conversion of the non-radioactive iodinated ryanodine analog to the [125I] isotope was accomplished by conversion of 7 to the trimethyltin derivative followed by [125I] exchange using chloramine-T in organic solvent. The radioiodinated ryanodine analog, 9, bound to cardiac membrane preparations in a protein dependent and saturable manner indicating that this analog may represent a useful new tool for the study of ryanodine receptors and that modifications about the C-10 hydroxy group of ryanodine may be carried out without loss in biological activity.

Animals↗