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, radio-iodinated 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.

Oxidative iodine monochloride iodination technique.

The iodine monochloride (IC1) technique is used to radiolabel proteins under mild experimental conditions. Proteins labeled by this technique have been shown to have both in vitro in vivo characteristics often superior to those proteins labeled by the more frequently used chloramine-T method. Although first published more than two decades ago, use of the IC1 technique has largely been superceded by other more recently developed iodination methods. This paper attempts to reintroduce th oxidative IC1 method to the research community. This is accomplished by the presentation of a detailed protocol for a convenient modification of the original technique. A review of the qualities of the iodinated proteins produced by the IC1 and other iodination methods is also discussed.

A microradioiodination method for detection of nanogram amounts of proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A procedure is described for the rapid analysis of nanogram quantities of protein resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Proteins are first radioiodinated by the chloramine T method, acid precipitated after addition of a visible marker protein and collected on a nitrocellulose filter. The region of the filter containing protein is excised and the proteins are then extracted from the filter into electrophoresis sample buffer.

Enzymatic labeling of gamma-globulin and insulin with iodine-125.

The parameters of enzymatic labeling of proteins with iodine 125 were examined. The manner and sequence of reagent addition, the effects of reagent concentration, reaction time and total Na125I activity on the labeling yield were determined.

Preparation of 125I-thyroxine and 125I-triiodothyronine of high specific activity for the radioimmunoassay of serum total T4 and T3.

A simple and inexpensive procedure for the preparation of high specific activity 125I radioiodinated triiodothyronine and thyroxine is described. The specific activities achieved are approximately 350 Ci/g for 125I-T4, and either 200 Ci/g for 125I-T3 depending on the reagents employed. The high specific activity 125I-T4 and 125I-T3 are suitable for the routine radioimmunoassay of serum total T3 and T4. When properly stored they have a self life of at least seven weeks.

Studies on experimental iodine allergy: 2. Iodinated protein antigens and their generation from inorganic and organic iodine-containing chemicals.

We hypothesize that iodine allergy is an immune response to iodinated autologous proteins generated in vivo from iodine-containing organic and inorganic chemicals. In this report, effects of protein iodination on elicitogenic activity in guinea pig iodine allergy model and iodinated protein antigen generation in vitro from iodine-containing chemicals were investigated. Active cutaneous anaphylaxis (ACA) and delayed-type hypersensitivity (DTH) tests were performed in guinea pigs immunized with iodine. The amount of iodine (I2) reacted to proteins for giving them an eliciting activity of ACA was > or = 0.15 micromol for 1 mg of albumin. DTH reactions were provoked by intradermal injection of 10(6) PECs reacted with > or = 0.075 micromol of I2. I2 was generated from a potassium iodide (KI) solution or iodinated contrast media by UV light irradiation. X-ray irradiation of KI and iodinated contrast media in the presence of protein resulted in the generation of iodinated protein antigens. The generation of iodinated protein antigens was inhibited in the presence of reducing agents. Therefore, it is noteworthy that iodine allergy of the present hypothesis is dependent on reactive oxygens. By presenting these ex vivo and in vitro data, we discuss the possibilities for the generation of iodinated protein antigens in vivo.

Characterization of the binding of human growth hormone to microsomal membranes from rat liver.

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 X 10(10) +/- 0.2 X 10(10)M-1 and 0.03 X 10(10) +/- 0.007 X 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.

Preparation and reactions of an iodinated imidoester reagent with actin and alpha-actinin.

The chemical iodination of an imidoester (methyl-p-hydroxybenzimidate, Wood et al. (1975) Anal. Biochem. 68, 339) and subsequent coupling of iodinated imidoester (IIE) to protein is an indirect method of iodinating proteins that is specific for the epsilon amino group of lysine residues and maintains the positive charge on the amino group at physiological pH. Purification of the IIE from chloramine-T and free iodine by benzene extraction eliminates the need for isoelectric precipitation and produces a more time- and cost-efficient IIE preparation and purification protocol. The separation of free from protein-bound label by chromatography, using centrifugal elution, provides a separation method that is rapid and efficient, without the generation of large volumes of radioactive wastes characteristic of conventional chromatographic and dialysis methods. To optimize the parameters of labeling protein with IIE, a systematic assessment of the effects of pH, reactant concentrations, and reaction time was made using purified cardiac actin and gizzard alpha-actinin. The parameters were defined to achieve an average labeling ratio of one IIE per protein polypeptide. The data demonstrate that both proteins appear to be labeled at the same rate and define several determining factors that limit the rate and extent of IIE incorporation into protein.

Formation of potential antigens from radiographic contrast media.

The use of radiographic contrast media is occasionally accompanied by more or less serious adverse effects, evidently of complex etiology, following intravascular administration. Some of these reactions are suspected of having an allergic basis. The in vitro and in vivo formation of iodinated serum proteins following gamma irradiation in the presence of two commonly used radiographic contrast media is demonstrated. Non-toxic concentrations of ascorbate present during the irradiation is shown to prevent the formation of such iodo-proteins in vitro as well as in vivo. The amounts of potentially antigenic iodoprotein formed during radiographic procedures will certainly be very small, but this quantity may be sufficient to elicit a hypersensitivity reaction in cases when an individual has been previously sensitized to immunologically similar iodo-proteins, a mechanism that could account for certain rare and unpredictable reactions. The radiation induced formation of iodo-proteins may also serve as a model for the formation of iodine containing antigens mediated by a free radical mechanism, i.e. in the metabolism of iodinated compounds like erythrosine, a widely used colouring agent for certain foods.