Iodination of staphylococcal enterotoxin B by use of chloramine-T.

This report describes the conditions that are necessary for iodination of staphylococcal enterotoxin B (SEB) by use of chloramine-T. Makor Chemical Co. SEB and the two major SEB components, which were prepared by isoelectric focusing of partially purified SEB, were used in these studies. The antigenic activity of the SEB preparations was monitored by radioimmunoassay as the oxidation/reduction (O/R) potential was increased by addition of chloramine-T. The SEB preparations lost antigenic activity rapidly at pH 7.5 and room temperature when sufficient chloramine-T was added to raise the O/R potential above 250 mV. Iodinated SEB with satisfactory immunoreactivity was prepared by omitting carrier iodide from the iodination reaction mixture and by using at least 1 mg of SEB/ml, steps which made the O/R potential more stable, and by stopping the reaction before the O/R potential exceeded 250 mV. Comparison of the chloramine-T method with a lactoperoxidase/H2O2 method of iodinating SEB showed the latter to cause a greater loss of immunoreactivity.     

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.     

Radioiodination of Enterotoxin from Clostridium perfringens Type A using Chloramine T

Procedures were examined for labelling enterotoxin isolated from Clostridium perfringens type A. with ¹²⁵I using chloramine T as the oxidizing agent. The iodination method was evaluated critically to establish the optimal conditions for the preparation of iodinated enterotoxin with a high specific radioactivity and without impairing the immunospecificity and biological activity. The use of 250 μg/ml of chloramine T in the reaction mixture. 500–1000 μCi of Na¹²⁵I/10 μg of enterotoxin and a reaction time of 40 s at pH 7–0 produced ¹²⁵I-enterotoxin of both high specific radioactivity and immunospecificity which retained its biological activity. No damage or aggregate formation due to the iodination process was observed. Enterotoxin labelled with high specific activity (135 μCi μg) showed extensive dissociation of ¹²⁵I when stored at 4°C and—20°C. In contrast, toxin labelled with low specific activity (7 μgCi/μg) was stable for as long as two months. The immunoreactivity of all labelled preparations was essentially unchanged after storage for one month.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in the sites of iodination of proteins following four methods of radioiodination

The rate of deiodination of radioiodinated proteins varies with the method of iodination. To elucidate differences in the iodinated protein labeled by various methods, we have hydrolyzed fibrinogen and several small peptides iodinated by the iodine monochloride, chloramine-T, electrolytic and enzymatic methods. Under conditions of either acidic or basic proteolysis, extensive deiodination occurred and the major product was I⁻. When a protease of Streptomyces griseus was used, radioiodinated fibrinogen and other polypeptides were degraded to single iodinated amino acid residues and only a small yield of I⁻. The iodinated amino acids resulting from proteolysis were separated by ion-exchange chromatography. The iodine monochloride and enzymatic methods yielded largely iodotyrosine with small amounts of other iodinated amino acids. The chloramine-T product spectrum varied with the chloramine-T:protein ratio, whereas the electrolytic method yield was a complex function of the reaction conditions. The different methods of iodination lead to some differences in the site of iodination which correlate with stability of the protein-iodine bond.     

The effect of oxidation of the Met27 residue of [125I]monoiodoglucagon on receptor-binding affinity

When glucagon is iodinated by the chloramine-T method, the Met27 residue is oxidized. This is not the case when the iodination is performed by the lactoperoxidase method. The two preparations can be purified to the same specific activity using QAE-Sephadex A-25 ion exchange chromatography. Receptor-binding studies in isolated rat adipocytes or hepatocytes revealed that the oxidized from possessed an average-binding affinity which is only about two thirds of that of the non-oxidized form. The reduced affinity of the oxidized tracer cannot be explained by an increased rate of dissociation.     

Isolation and hydroxylysine glycoside content of some cyanogen bromide-cleaved fragments of collagen from bovine corneal stroma.

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.     

Iodinated derivatives of vasoactive intestinal peptide (VIP), PHI and PHM: purification, chemical characterization and biological activity

The iodination of vasoactive intestinal peptide (VIP) was studied, using a variety of enzymatic and chemical iodination methods. Reversed phase high performance liquid chromatography (HPLC) was used to purify the reaction products. The lactoperoxidase-glucose oxidase method gave excellent results in terms of reproducibility, iodine incorporation, and yield of the non-oxidized products [Tyr(I)10]VIP and [Tyr(I)22]VIP, and was used to prepare both 125I and 127I labelled derivatives. In both cases, direct application to HPLC and a single column system were used. Although the oxidized peptides [Tyr(I)10,Met(O)17]VIP and [Tyr(I)22,Met(O)17]VIP could be generated to varying degrees directly by iodination of VIP, these were most conveniently prepared by iodination of [Met(O)17]VIP. Iodinated derivatives of the homologous peptides PHI and PHM were likewise prepared by rapid, one-step HPLC procedures. The site and degree of iodination were determined by HPLC peptide mapping of tryptic digests and amino acid analyses, and in the case of [Tyr(I)10]VIP also by sequencing. The vasorelaxant activities of the iodinated peptides in bovine cerebral artery preparations did not differ significantly from those of the corresponding noniodinated peptides, with the exception of [Tyr(I)10,Met(O)17]VIP and [Tyr(I)22,Met(O)17]VIP which, unlike [Met(O)17]VIP itself, had slightly lower potency than VIP.     

Biological activity of iodinated gastrins.

Mono- and di-iodo heptadecapeptide gastrins were prepared by a modified chloramine-T technique. The iodinated gastrins did not differ significantly from unmodified gastrins in immunoreactivity and in stimulating acid secretion in gastric fistula dogs. The preparations should prove useful in studies of hormone binding to receptors.     

Preparation and immunoreactive properties of monoiodinated angiotensin labelled at high specific activity

Monoiodinated angiotensin II at a specific activity in the order of 1 C/mg can be prepared with high yields by controlling the pH of iodination and can be purified on G-25 Sephadex. The fractions from the head to the centre of the single peak of radioactivity obtained by gel filtration of the iodination mixture contain pure monoiodinated angiotensin. The monoiodinated derivative shows an immunoreactivity very close to that of the native hormone; in contrast, the diiodinated derivative has a low immunoreactivity and its use as a tracer results in a loss of sensitivity in the radioimmunoassay of angiotensin II. It is suggested that the use of the monoiodinated derivative in pure form should be recommended whenever a small hormonal polypeptide labelled at high specific activity is to be prepared for radioimmunoassay purposes.     

A mechanism and an evaluation of surface specific iodination by the chloramine-T procedure.

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.     

Kinetics of redox interaction between substituted quinones and ascorbate under aerobic conditions.

One-electron reduction of quinones (Q) by ascorbate (AscH ); (1) AscH + Q --> Q*- + Asc*- + H+, followed by the oxidation of semiquinone (Q*-) by molecular oxygen; (2) Q*- + O2 --> Q + O2*-, results in the catalytic oxidation of ascorbate (with Q as a catalyst) and formation of active forms of oxygen. Along with enzymatic redox cycling of Q. this process may be related to Q cytotoxicity and underlie an antitumor activity of some Qs. In this work, the kinetics of oxygen consumption accompanied the interaction of ascorbate with 55 Qs including substituted 1,4- and 1,2-benzoquinones, naphthoquinones and other quinoid compounds were studied in 50 mM sodium phosphate buffer, pH 7.40, at 37 degrees C by using the Clark electrode technique. The capability of Q to catalyze ascorbate oxidation was characterized by the effective value of kEFF calculated from the initial rate of oxygen consumption (R(OX)) by the equation R(OX) = kEFF[Q][AscH-] as well as by a temporary change in R(OX). The correlation of kEFF with one-electron reduction potential, E(Q/Q*-), showed a sigma-like plot, the same for different kinds of Qs. Only the Qs which reduction potential E(Q/Q*-) ranged from nearly -250 to + 50 mV displayed a pronounced catalytic activity, kEFF increased with shifting E(Q/Q*-) to positive values. The following linear correlation between kEFF (in M (-1) s(-1)) and E(Q/Q*-) (in mV) might be suggested for these Qs: lg(kEFF)= 3.91 + 0.0143E(Q/Q*-). In contrast, Qs with E(Q/Q*-) < - 250 mV and E(Q/Q*-) > + 50 mV showed no measurable catalytic activity. The Qs studied displayed a wide variety in the kinetic regularities of oxygen consumption. When E(Q/Q*-) was more negative than - 100 mV, Q displayed a simple ('standard') kinetic behavior--R(OX) was proportional to [AscH-][Q] independently of concentration of individual reagents, [AscH-] and [Q]; R(OX) did not decrease with time if [AscH-] was held constant: Q recycling was almost reversible. Meanwhile, Qs with E(Q/Q*-) > - 100 mV demonstrated a dramatic deviation from the 'standard' behavior that was manifested by the fast decrease in R(OX) with time and non-linear dependence of even starting values of R(OX) on [Q] and [AscH-]. These deviations were caused basically by the participation of Q*- in side reactions different from (2). The above findings were confirmed by kinetic computer simulations. Some biological implications of Q-AscH- interaction were discussed.     

Enzymatic radioiodination of succinyl cyclic AMP tyrosine methyl ester by lactoperoxidase and radioimmunoassay for cyclic AMP.

A highly specific and simple radioimmunoassay for cyclic AMP with a sensitivity of 0.04 picomoles/tube has been developed according to the method of Steiner et al., using 125I-succinyl cyclic AMP tyrosine methyl ester as a tracer. The tracer with higher immunoactivities could be simply and constantly prepared by an enzymatic iodination procedure utilizing lactoperoxidase, radioactive iodide and hydrogen peroxide generated by glucose-glucose oxidase system, rather than by chloramine-T procedure.     

Iodination of biological samples without loss of functional activity

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.     

Effect of iodination on the activity of cytotoxin P4 of Naja nigricollis nigricollis.

Iodination of cytotoxin P4, isolated from the venom of Naja nigricollis nigricollis, develops gradually and depends on the molar ratio between the free iodine and the cytotoxin reaching a maximum of two equivalents at a molar ratio of 250 or higher. The cytotoxic activity was also gradually decreased and was totally abolished when one equivalent of iodination was achieved. However, antigenic properties of the cytotoxin were preserved in the iodinated form. When the iodination of the cytotoxin was carried out with a carrier free radiolabeled iodide, the molar ratio was 0.05 resulting in labelling of only 2% of the cytotoxin molecules, which explains the cytotoxicity of the radiolabeled mixture.     

Evaluation of iodovinyl antibody conjugates: comparison with a p-iodobenzoyl conjugate and direct radioiodination

The preparations and conjugations of 2,3,5,6-tetrafluorophenyl 5-[125I/131I]iodo-4-pentenoate (7a) and 2,3,5,6-tetrafluorophenyl 3,3-dimethyl-5-[125I/131I]iodo-4-pentenoate (7b) to monoclonal antibodies are reported. Reagents 7a and 7b were prepared in high radiochemical yield by iododestannylation of their corresponding 5-tri-n-butylstannyl precursors. Radioiodinated antibody conjugates were prepared by reaction of 7a or 7b with the protein at basic pH. Evaluation of these conjugates by several in vitro procedures demonstrated that the radiolabel was attached to the antibody in a stable manner and that the conjugates maintained immunoreactivity. Comparative dual-isotope biodistribution studies of a monoclonal antibody Fab fragment conjugate of 7a and 7b with the same Fab fragment labeled with N-succinimidyl p-[131I]iodobenzoate (PIB, p-iodobenzoate, 2) or directly radioiodinated have been carried out in tumor-bearing nude mice. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 2 demonstrated that the biodistributions were similar in most organs, except the neck tissue (thyroid-containing) and the stomach, which contained substantially increased levels of the 7a label. Coinjection of the Fab conjugate of 7a with the Fab fragment radioiodinated by using the chloramine-T method demonstrated that the biodistributions were remarkably similar, suggesting roughly equivalent in vivo deiodination of these labeled antibody fragments. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 7b indicated that there was approximately a 2-fold reduction in the amount of in vivo deiodination of the 7b conjugate as compared to the 7a conjugate.     

Identification of antigenic sites on staphylococcal enterotoxin B and toxoid

The staphylococcal enterotoxins (SEs) are capable of causing both food poisoning and a toxic shock-like illness in man. In addition, SEs are known to act as superantigens, stimulating T-cells according to their T-cell receptor Vβ type. Relatively little is known of their antigenic determinants and how these may relate to the structure and function of the toxins. As a step in the study of these relationships, the entire molecule of SEB was synthesized in duplicate as a series of octapeptides overlapping by seven residues. This series thus represented all the potential linear epitopes of eight residues or less. The reactivity of the octapeptide series with antisera raised to purified SEB and to formaldehyde-inactivated SEB has been used to locate several antigenic sites on native SEB and to identify antigenic differences in the toxoid. Three antigenic peptides identified from the antigenic profile were synthesized and characterized. These represented amino acids 21–32, 93–107 and 202–217 of SEB. None of these peptides affected SEB-induced T-cell proliferation. However, the occurrence or absence of cross-reactivity of these peptides with antibodies to native SEB corresponds to the degree of exposure and/or the rigidity of these regions within SEB.     

Single-step purification of recombinant human growth hormone (hGH) directly from bacterial osmotic shock fluids, for the purpose of (125)I-hGH preparation

A good quality tracer, to be used in the radioimmunoassay of human growth hormone, was prepared by applying the chloramine-T iodination technique to the recombinant product obtained after a single-step high-performance size-exclusion chromatography purification of a bacterial osmotic shock fluid. The labeling reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of approximately 50% (NIDDK reference antiserum diluted 1:600,000). These values are very similar to those obtained by radioiodinating highly purified clinical-grade recombinant human growth hormone obtained from the same periplasmic extract after the regular six-step purification process. Both tracers provided the same accuracy, when evaluated with the use of commercial-quality control samples in a classical radioimmunoassay methodology, their stability being practically identical: about 18% decrease in antibody binding after 2 months of storage at -20 degrees C. The novel approach permits the utilization of transformed Escherichia coli strains as a source of freshly prepared, radioiodination-grade recombinant proteins, capable of providing better reproducibility and reagent continuity.     

Radioimmunoassay for secretin using Nalpha-tyrosylsecretin and [Tyr1]-secretin.

Sensitive radioimmunoassay for secretin was developed by using synthetic preparation of porcine secretin and its related analogs. The secretin-specific antisera with titers ranging 1: 20,000-1 : 150,000 were generated in rabbits against highly purified synthetic secretin. The labeled antigen was prepared by radioiodinating by the chloramine-T method synthetic secretin analog, Nalpha-tyrosylsecretin or [Tyr1]-secretin, both of which were proved to have almost identical immunoreactivities with that of secretin itself. The immunoassay was performed by the double-antibody method using synthetic secretin as standard. The lowest detectable amount of secretin in the present assays was 5-10pg/tube. Human duodenum extract with hot water contained secretin or secretin-like material that shows a parallel displacement curve to the standard in the immunoassay system used. Serum levels of secretin immunoreactivity in man rose up to 250 pg/ml by intraduodenal infusion of HCl and to 800-1,000 pg/ml by i.v. injection of 1 cu/kg of Boots natural secretin.     

Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol

Mercaptoethanol, when present in the sample buffer during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is responsible for the appearance of two nonprotein bands (electrophoretic mobilities corresponding to 68 and 54 kdalton) stainable with silver and with Coomassie blue. After iodination in vitro of DNA preparations isolated by alkaline phenol extraction using chloramine-T procedure, part of the radioactive label is found in these bands, provided the reaction is terminated by mercaptoethanol, whereas only a diffuse background is present in this area if the reaction is stopped by sodium metabisulfite. Similar results are obtained with highly purified total cytoplasmic RNA. The results indicate that the appearance of the 68- and 54-kdalton bands is in artifact. The relevance of these results to the proteins tightly bound to DNA is discussed.