共查询到20条相似文献,搜索用时 15 毫秒
1.
De Spiegeleer B Van Dorpe S Vergote V Wynendaele E Pauwels E Van De Wiele C Garcia-Solis P Solis-Sainz JC 《Peptides》2012,33(2):272-278
Different iodinated mouse obestatin peptides have been characterized toward their in vitro stability in the main metabolic compartments plasma, liver and kidney. Using HPLC-UV for quantification, significant differences in the degradation kinetics of the iodinated peptides, arising from both enzymatic proteolysis and dehalogenation, were found when compared to the native, unmodified peptide. HPLC-MS/MS analysis demonstrated that the cleavage sites were dependent upon the biological matrix and the location of the amino acid residue incorporating the iodine atom(s). The degrading proteases were found to target peptide bonds further away from the iodine incorporation, while proteolytic cleavages of nearby peptide bonds were more limited. Diiodinated amino acid residue containing peptides were found to be more susceptible to deiodination than the mono-iodinated derivative. In plasma, the percentage of peptide degradation solely attributed to deiodinase activity after 20 min incubation reached up to 25% for 2,5-diiodo-H(19)-obestatin compared to 20% and only 3% for (3,5-diiodo-Y(16))- and (3-iodo-Y(16)) obestatin, respectively. Hence, our results demonstrate that the different iodinated peptides pose significantly different metabolization properties and thus, also different biological activities are expected for peptides upon iodination. 相似文献
2.
Chemical and enzymatic modification of proteins in the 30 S ribosome of Escherichia coli 总被引:1,自引:0,他引:1
Methods are described for the iodination of ribosomal proteins by iodine monochloride and potassium iodide and bovine lactoperoxidase. Ribosomes that were maximally iodinated did not synthesize polyphenylalanine. About one-half of the tyrosine residues could be iodinated with iodine monochloride in the intact ribosome with no change in the sedimentation properties of the particle. When proteins were extracted and dissolved in 5 m-urea, all of the tyrosine residues could be iodinated with iodine monoehloride. 相似文献
3.
A technique for radioactive labeling by iodination of sensitive biological material that preserves the functional activity of the samples to a greater extent than the standard iodination methods is presented. The reaction is carried out keeping in separate phases the substrate and the iodine-generating system. Chloramine-T in the presence of water and Cl? ions generates Cl2 on a piece of filter paper kept close to the surface of the solution containing the substrate and the 125I?. The Cl2 molecules diffuse from the paper, enter the solution, and, reacting with the iodide ions, generate iodine that modifies the aromatic rings of the sample. Protein A, lysozyme, and ribosomes treated under these iodination conditions are much less affected in their activity than when iodinated by the standard chloramine-T method or the iodogen system. In addition, this technique, which we have called “two phases” system, seems to act preferentially on the surface of the structures as shown by studying the iodination pattern of the ribosomal proteins. 相似文献
4.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1988,15(6):701-706
The monoclonal antibody (MAb) 155H.7, raised against a synthetic β-anomer of the Thomsen-Friedenreich antigen (S-TAG), was radioiodinated using iodine monochloride, chloramine-T and Iodogen and radiolabeled with 111In using the bromoacetamido-derivative of benzyl-EDTA. The in vitro immunoreactivity of the MAb was assessed using an ELISA with the S-TAG and the in vivo distribution of the radioiodinated and radiochelated MAb was determined in the murine mammary carcinoma TA3/Ha tumour model. Both chloramine-T and iodine monochloride radioiodination greatly reduced the immunoreactivity of the MAb compared to radioiodination using lodogen. Bifunctional chelate labeling was comparable to Iodogen in reducing the immunoreactivity of the MAb and subsequent chelation of 111In did not further compromise the immunoreactivity of the MAb. The in vivo distribution data showed significantly different distributions of the radiolabels after injection of the radioiodinated and radiochelated MAb. The 131I-MAb showed some tumour association as compared to the distribution of an 125I-non-specific protein and the data also indicates that there is preferential dehalogenation of the radioiodinated MAb. 111In from the radiochelated MAb showed significantly higher uptake in the tumour than 131I from the 131I-MAb. It is suggested that the differing fates of the two radiolabels within the tumour cell is responsible for the difference in retention observed and not necessarily due to the lack of MAb uptake by the tumour. Overall, the radiochelate label for MAb 155H.7 appears to be superior to radioiodine for in vivo use. 相似文献
5.
Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril 总被引:53,自引:0,他引:53
Films of 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril (conveniently “plated” in reaction vessels from methylene chloride solution) react rapidly in the solid phase with aqueous mixtures of I? and proteins to yield iodinated proteins. Similarly applied, this reagent brings about iodination of cell membranes, although, apparently, at a somewhat lesser rate than in iodinations carried out with lactoperoxidase-glucose oxidase. The stability and sparing solubility of this chloroglycoluril in water can account for the minimal damage to proteins and living cells observed in these iodinations; further, these properties allow for elimination of the reduction step employed at the close of iodinations with soluble chloroamides such as chloramine-T. 相似文献
6.
Solid-phase methodology has previously been applied to labeling of proteins and peptide hormones used in immunoassay with the aid of enzyme sorbent. In this publication a method based on the use of a new carrier-copolymer of maleic anhydride and butanediol divinylether is introduced. As a model, bovine serum albumin (BSA) was labeled using three different procedures: Chemical, with chloramine-T as oxidizing agent: enzymatic, in a liquid phase with lactoperoxidase (LP) and horseradish peroxidase (HRP); and enzymatic, in a solid phase with maleic anhydride butanediol divinylether-copolymer as the carrier of lactoperoxidase and horseradish peroxidase.The lactoperoxidase-mediated iodinating activity in both the liquid and solid phases was similar (incorporating 47 and 39% for the total 125iodine added, 1 ), while HRP was more efficient in a liquid (11%) than in a solid phase (3%).Although the specific activity of the BSA labeled with chloramine-T was highest, this 125I-labeled BSA was badly degraded during iodination. However, in either liquid or solid phase enzymatic iodinations, no degradation of the protein could be observed.Peptide hormones, luteinizing hormone, follicle-stimulating hormone and angiotensin II, iodinated with lactoperoxidase or lactoperoxidase sorbent for radioimmunoassays reacted better than peptide hormones iodinated with chemical oxidants and remained unaltered during storage. 相似文献
7.
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. 相似文献
8.
G L De Nardo S J De Nardo N P Miyao S L Mills J S Peng L F O'Grady A L Epstein W C Young 《The International journal of biological markers》1988,3(1):1-9
In patients or mice with cancer the pharmacokinetic behavior of radioiodinated and radiometal chelated antibodies has been observed to be different. Rapid clearance from the tissues and excretion into the urine can occur after injection of radioiodinated antibodies. These observations have been interpreted to reflect in vivo dehalogenation of the antibody. This publication describes a variety of other mechanisms that can underlie these phenomena. These mechanisms include receptor uptake and catabolism of antibody and instability of the labeled antibody due to the labeling conditions. Specifically, the relative masses of chloramine-T and antibody in the iodination reaction mixture, the level of iodination of the antibody, and the amount of antibody administered to the recipient are all factors which can influence the clearance of radioiodinated antibody from the recipient. The final determinant for the different behavior of radioiodinated and In-111 metal chelated antibody relate to the different biologic pathways of indium when compared to iodine. 相似文献
9.
A previously reported method for iodination of the tyrosine moiety of oxidation-sensitive biomolecules was found to cause unacceptable damage to biomolecules containing thiols and thioether groups. This was due to the oxidation of the sulfur-containing residues by molecular iodine (I(2)). To selectively iodinate the tyrosine moiety with minimum oxidation to the sulfur functionality, studies of the kinetics of the reactions between I-(3) and various amino acids and small peptides at various pH values in phosphate buffer were undertaken. Within the pH range studied (5.5-8.2), the results showed that the iodination reaction is strongly catalyzed by hydroxide ions, whereas the oxidation of the sulfur group was insensitive to pH. The results also showed that both reactions are strongly catalyzed by HPO-(4) ion. In a complex molecule, such as methionine-enkephalin, oxidation of the methionine residue (undesirable reaction) proceeds in parallel with iodination of the tyrosine residue (desirable reaction). If such a molecule was iodinated in 0.01 M phosphate buffer at pH values above 7.5, the iodination reaction would proceed much more rapidly than the oxidation reaction, resulting in a high yield of iodinated substrate with little oxidative damage. 相似文献
10.
Self-association and phospholipid binding properties of iodinated apolipoprotein A-I 总被引:2,自引:0,他引:2
Kinetic turnover studies of apolipoprotein metabolism often utilize radioiodinated tracers. These studies rely on the "tracer assumption" that the modified tracer is physiologically and metabolically identical with the native unmodified tracer. This paper addresses the validity of this assumption on the basis of the examination of the state of self-association and binding properties with egg yolk phosphatidylcholine small unilamellar vesicles of native and iodinated apolipoprotein A-I (apoA-I). Human apoA-I was iodinated to the extent of 1.0 and 3.7 mol of nonradioactive iodine/mol of protein. At concentrations from 0.013 to 0.8 mg/mL, iodinated apoA-I underwent concentration-dependent self-association similar to that of native apoA-I as evidenced by circular dichroism and gel filtration. At all concentrations, however, the iodinated preparations were more highly self-associated as judged by gel filtration in relation to the extent of iodination. Scatchard analysis of fluorometric titrations of apoA-I/vesicle interactions demonstrated that the binding capacity of vesicles for apoA-I increased and apoA-I binding affinity decreased upon iodination. In addition, the kinetics of apoA-I binding to vesicles was enhanced by iodination. The affinity, capacity, and kinetics of apoA-I binding were each altered 2-3-fold dependent on the extent of iodination. Since the dynamic interactions of apoA-I are perturbed by iodination, one may legitimately question whether the "tracer assumption" is valid for 125I-apoA-I under all experimental conditions. 相似文献
11.
Carol Dziadik Turner Steven B. Chernoff Alvin Taurog Allen B. Rawitch 《Archives of biochemistry and biophysics》1983,222(1):245-258
The distribution of iodine among the polypeptides of human goiter thyroglobulin (Tg) was examined. Tg was iodinated in vitro with 131I to levels of 2 to 84 gram atoms (g.a.)/mol using thyroid peroxidase (TPO) or a chemical iodination system. The samples were reduced, alkylated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two low-molecular-weight peptides appeared preferentially in radioautograms of the sodium dodecyl sulfate (SDS) gels of TPO-iodinated samples. Iodination of these peptides increased sharply in the TPO-treated Tg as the level of total iodine/ molecule rose. Radioiodine was incorporated into these same gel regions in the chemically treated Tg, but only after much higher levels of total iodination were reached. Differences in iodoamino acid distribution were also noted between the chemically and enzymatically iodinated thyroglobulins. In the chemically iodinated samples, little thyroxine (T4) was synthesized, even at high iodine levels. In the TPO-treated samples only small amounts of T4 were seen below 14 g.a. total I/mol, while at or above that level of iodination T4 formation increased sharply. To examine the coupling process, Tg was chemically iodinated, excess I? removed, and the samples treated with TPO and a H2O2-generating system in the absence of iodide. Radioautograms obtained from SDS-polyacrylamide gels of reduced and alkylated protein from such coupling assays showed an increase in the level of iodine in the low-molecular-weight peptides after TPO treatment. Thyroxine production also increased with TPO treatment. The addition of free DIT (a known coupling enhancer) to the [131I]Tg/TPO incubation increased both the production of T4 and the amount of iodine in the smaller polypeptides. Two-dimensional maps prepared from CNBr-digested TG showed differences between the coupled and uncoupled samples. Our observations confirm the importance of the lowmolecular-weight peptides derived from Tg in thyroid hormone synthesis. At total iodine levels above 14 g.a./mol Tg in enzymatically treated samples there is selective incorporation of iodine into both the low-molecular-weight polypeptides and into thyroid hormone. 相似文献
12.
Studies on the iodination of LH-RH and the biological and immunological activities of the products 总被引:2,自引:0,他引:2
Synthetic LH-RH was iodinated by the modified chloramine-T or lactoperoxidase method, using 127INa or 125INa. The yields of the products, the LH releasing activities of the monoiodinated peptides as well as binding to pituitary membrane fractions were measured. The variation in yield in the four procedures used for iodination was a function of the amount of oxidizing agent. Monoiodinated products obtained by the different procedures possessed comparable LH-releasing activities as well as binding affinity to pituitary plasma membranes. 相似文献
13.
1. The influence of carrier iodide, iodine monochloride and pH on the labelling of ox insulin with 131I by the iodine monochloride method have been studied. 2. The quantitative effect of the iodide in the radioactive iodine preparation was that predicted from a calculation of its specific activity. No other interfering factors were detected in the [131I]iodide solutions used. 3. Increasing the molar ratio of iodine monochloride to insulin resulted in an increase followed progressively by a decrease in the proportion of 131I bound, while the total iodine bound increased to an amount characteristic of pH and thereafter remained constant. 4. The influence of pH on the iodination of insulin with iodine monochloride was complex and the pH curve showed two maxima, at pH2·8 and 6·4. At pH2·8 it was not possible to exceed 8 atoms of iodine bound per molecule by increasing the molar ratio of iodine monochloride. Similarly, at pH6·4 the substitution value of 11·5 atoms of iodine per molecule could not be exceeded. 5. Iodinated insulins containing an average of 1·96, 2·74, 6·0 and 7·0 atoms of iodine per molecule fully retained the ability to bind guinea-pig anti-(ox insulin) serum, and the ability to compete with unlabelled insulin for antibody sites only became significantly changed in the most highly substituted preparations and in the presence of large concentrations of unlabelled insulin. 6. The method for the iodination of insulin with 98% incorporation of 131I by using chloramine-t is described. 7. 131I-iodinated insulin prepared with graded quantities of chloramine-t in excess of that required for efficient labelling was less efficiently bound by guinea-pig anti-(ox insulin) serum than insulin labelled by the iodine monochloride method. 相似文献
14.
Both internal and external proteins in vesicular stomatitis virus were labeled when intact virions were iodinated with 50 μm iodide; however, only the surface proteins were labeled when the same procedure was carried out at low iodide concentrations (below 0.5 μm). This result together with similar observations reported earlier with another enveloped virus, Rous-associated virus-61 (RAV-61), suggest that viral envelopes provide a barrier to iodination by chloramine-T at low, but not at high, iodide concentrations. By monitoring the permeability of the RAV-61 envelope to successive iodinations and to iodination in the presence of chaotropic thiocyanate ions, it was shown that the permeability of the viral envelope was not altered at the higher concentrations of iodide. Further results support the hypothesis that iodination mediated by chloramine-T inolves two different iodinating species: (a) a membrane impermeable one, possibly “iodamine-T,” which predominates at low iodide concentrations, and (b) a membrane permeable species, possibly molecular iodine, which predominates at high concentrations of iodide. These results reinforce the proposal that the chloramine-T procedure is a useful method for specifically labeling surface proteins of lipid-enveloped structures. 相似文献
15.
R.Arlen Rosenberg S.A. Muzaffar J.N.M. Heersche D. Jez T.M. Murray 《Analytical biochemistry》1983,128(2):331-341
A constant-current microelectrolytic radioiodination method was used to label bovine parathyroid hormone (BPTH) with 125I to an overall iodination ratio of 1:1 iodide atoms per PTH molecule. Such iodinated preparations were shown to be fully active in several bioassay systems: in vitro adenylate cyclase activation in rat renal and skeletal membranes, in vitro calcium release from rat calvaria, and the in vivo hypercalcemic response in chickens. Analysis by Sephadex G-15 chromatography after enzymatic digestion showed the radioiodine to be incorporated predominantly as monoiodotyrosine. Bioassay of iodinated preparations from which uniodinated hormone had been removed by isoelectric focusing showed essentially full hormonal activity. Such methods can be used to consistently produce radioiodinated biologically active preparations of BPTH 1–84 with high specific activity (2000 Ci/mmol). 相似文献
16.
A study of those tyrosines in fibrinogen which are surface-oriented and which may be involved in polymerization has been investigated using as a probe iodination catalyzed by lactoperoxidase. The iodine distribution in the major cyanogen bromide fragments was studied. A fragment of the B beta chain extending beyond residue 118 had the highest specific activity. Tyrosine 119 was identified as the residue most susceptible to iodination. There was no difference in susceptibility to iodination of N-DSK (A alpha 1-51, B beta 1-118, gamma 1-78)2, Ho1-DSK (first hydrophobic disulfide knot), and Hi2-DSK (second hydrophobic disulfide knot). Tyrosines 18 and 32 of the gamma chain of N-DSK were not significantly iodinated in fibrinogen, but tyrosines 1 and 68 were labeled, as was the tyrosine of the A alpha chain. The data indicate that there are regions of the hydrophobic disulfide knot, Ho1-DSK, which are surface-oriented. The distribution of iodine as mono- and diiodotyrosine in N-DSK and Ho1-DSK reflected the percentage (83 and 17, respectively) found in iodinated fibrinogen from which these fragments were prepared. In contrast the segments of the B beta chain extending beyond Met118 contained 46% of the iodine in diiodotyrosine, while the A alpha chain fragment, Hi2-DSK, contained 28% as diiodotyrosine. No significant iodination of histidine was detected. 相似文献
17.
Isolation and hydroxylysine glycoside content of some cyanogen bromide-cleaved fragments of collagen from bovine corneal stroma. 总被引:1,自引:0,他引:1
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1. Guinea-pig low-density lipoproteins were isolated by ultracentrifugation and iodinated either by the IC1 method or by the chloramine-T procedure. 2. The efficiency of labelling by both methods was essentially the same. 3. When the two products were compared by ultracentrifugation, gel chromatography and immunodiffusion analysis, no significant difference in their properties was detected. 4. When they were compared by gradient-gel electrophoresis, aggregates were found in the product of the IC1 method, but not in the lipoprotein iodinated by the chloramine-T process. 5. Both products were metabolized by the guinea pig with essentially the same fractional catabolic rate. 6. The fractional catabolic rate of lipoprotein iodinated by the chloramine-T method was not significantly different from that of lipoprotein biologically labelled in the protein moiety with [75Se]selenomethionine. 7. It is concluded that the products of both methods of iodination are almost equally acceptable, provided that the optimum conditions for the chloramine-T reaction are carefully established. 相似文献
18.
M. Schubert J. Engel L. Th?ny-Meyer F. W. M. R. Schwarze J. Ihssen 《Applied and environmental microbiology》2012,78(20):7267-7275
In the present work, Norway spruce wood (Picea abies L.) was reacted with a commercial Trametes versicolor laccase in the presence of potassium iodide salt or the phenolic compounds thymol and isoeugenol to impart an antimicrobial property to the wood surface. In order to assess the efficacy of the wood treatment, a leaching of the iodinated and polymerized wood and two biotests including bacteria, a yeast, blue stain fungi, and wood decay fungi were performed. After laccase-catalyzed oxidation of the phenols, the antimicrobial effect was significantly reduced. In contrast, the enzymatic oxidation of iodide (I−) to iodine (I2) in the presence of wood led to an enhanced resistance of the wood surface against all microorganisms, even after exposure to leaching. The efficiency of the enzymatic wood iodination was comparable to that of a chemical wood preservative, VP 7/260a. The modification of the lignocellulose by the laccase-catalyzed iodination was assessed by the Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) technique. The intensities of the selected lignin-associated bands and carbohydrate reference bands were analyzed, and the results indicated a structural change in the lignin matrix. The results suggest that the laccase-catalyzed iodination of the wood surface presents an efficient and ecofriendly method for wood protection. 相似文献
19.
Alexander Benenson Marcel Mersel Arié Pinson Michael Heller 《Analytical biochemistry》1980,101(2):507-512
Phospholipids were iodinated with iodide by a lactoperoxidase-catalyzed reaction in the presence of controlled amounts of H2O2 which were continuously supplied by glucose oxidase + glucose. Different molecular and ionic species of inorganic iodine present in the reaction mixture (i.e., I?, I2, I3?) were eliminated by thiosulfate reduction to I? followed by gel filtration on Sephadex LH-20 which separated I? from the phospholipids completely. Final separation and identification of individual phospholipids were done on a column of silica gel H using a single solvent mixture consisting of CHCl3:CH3OH:CH3COOH:H2O (25:15:4:2, by volume). Application of phospholipases A2 and D or transesterification provided evidence to indicate a covalent iodination of the fatty acid moiety of the lipids by the enzymatic process, which apparently is substitution but could also proceed by addition to the double bonds, when present. 相似文献
20.
P C Srivastava D J Buchsbaum J F Allred P G Brubaker D E Hanna J K Spicker 《BioTechniques》1990,8(5):536-545
A new radioiodinating agent, N-(p-[125I]iodophenyl)maleimide, has been synthesized for its potential utility in the radioiodination of monoclonal antibodies and other proteins. The efficiency of incorporation of 125I in the B72.3 antibody by iodine monochloride iodination or by maleimide conjugation was 19% and 43%, respectively. The thyroid uptake following intraperitoneal administration of the two 125I-labeled antibody preparations was evaluated in nude mice implanted with LS174T colon carcinoma xenografts. The iodine monochloride preparation showed substantially greater uptake in the thyroid with values of 2.1% ID at 6 h after injection and reaching a maximum of 4.3% ID after 6 days. In contrast, the maleimide preparation showed a uniformly low uptake in thyroid of 0.1%-0.2% ID. The results of these preliminary studies demonstrate that the N-(p-[125I]iodophenyl)maleimide-labeled monoclonal antibodies showed markedly less (less than 10-fold) uptake of radioiodine in the thyroid, indicating significantly less in vivo deiodination than iodine monochloride-labeled monoclonal antibodies, while retaining some tumor localization in vivo. Studies are in progress to optimize N-(p-[125I]iodophenyl)maleimide radioiodination conditions and to improve the tumor localization. 相似文献