Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.     

NADPH oxidation catalyzed by the peroxidase/H2O2 system. Iodide-mediated oxidation of NADPH to iodinated NADP

Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280-nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.     

Conformation of pepsin and pepsinogen: some aspects of the role of tyrosine residues and the 1-44 segment of pepsinogen on conformational stability

Conformational changes induced in pepsin and pepsinogen by iodination of tyrosine residues and the possible role of lysine residues on conformational stability of pepsinogen are investigated by circular dichroism (CD) studies in solution. At low degrees of iodination (6 I/molecule) the pepsin molecule denatured, with complete loss of β-structure at pH 5.5. Pepsinogen showed greater resistance to conformational change on iodination (10 I/molecule) and about 30% of its ordered structure is retained. In the aromatic region, the tyrosyl CD bands of iodinated pepsin decreased in intensity, indicating a change in the environment of tyrosine residues. A comparison with the CD spectra of expanded structures of pepsin in 6 m guanidine hydrochloride or alkaline solutions (pH 9.75) indicated retention of a significant amount of tertiary structure in iodinated pepsin. Changes in tertiary structures were marginal on iodination of pepsinogen. Less than 1% (residue moles) of poly-l-lysine, a known inhibitor, was found to destabilize the secondary and tertiary structure of pepsin at pH 6.75, although the lysine-rich 1–44 segment of pepsinogen tends to stabilize the conformation of the pepsin chain. This seems to suggest that the inhibitory effects of polylysine on pepsin occur by a mechanism different from that of the activity-limiting effect of the lysine-rich 1–44 segment of pepsinogen.     

Iodinated derivatives of the hormone avian pancreatic polypeptide

Reaction of avian pancreatic polypeptide with an iodine monochloride reagent at both pH 4 and pH 7.5 results in the differential modification of the four tyrosine residues in this peptide hormone. A total of 19 distinct iodinated derivatives were isolated by reverse-phase high-performance liquid chromatography, and their sites of iodination were characterized by both tryptic mapping and leucine aminopeptidase techniques coupled with HPLC. The pH 4 reaction produced 16 derivatives which, overall, represented substantial iodination at each tyrosine residue, whereas the pH 7.5 reaction was more directed, producing only 7 derivatives. Iodination at the C-terminal tyrosineamide 36 predominated at both pH values, and diiodo-Tyr 36 was found in the majority of the pH 7.5 derivatives. The relative of the four tyrosine residues with ICl were as follows: at pH 7.5, Tyr 36 much greater than Tyr 21 much greater than Tyr 27 greater than Tyr 7; at pH 4, Tyr 36 greater than Tyr 27 greater than Tyr 7 greater than Tyr 21.     

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.     

The binding sites of insulin-like growth factor I (IGF I) to type I IGF receptor and to a monoclonal antibody. Mapping by chemical modification of tyrosine residues

The surface topography of IGF I(insulin-like growth factor I) was investigated by chemical modification of amino acid residues in free IGF I and bound to type I IGF receptor or to monoclonal antibody MAB43. Tyrosine residues were modified either by chloramine-T or lactoperoxidase catalyzed iodination. In the free IGF I molecule, all 3 tyrosine residues, A19 (Tyr-60), B25 (Tyr-24), and C2 (Tyr-31), were iodinated. Monoclonal antibody MAB43 protected IGF I against modification at tyrosine residue A19, and in the type I IGF receptor-IGF I complex, all 3 tyrosine residues were shielded against iodine incorporation. These results allow the prediction of the binding domains in the IGF I molecule. The minimal receptor binding site in IGF I would include amino acid residues B25 to C2 and, possibly, the C-terminal part of the A-domain with tyrosine residue A19.     

Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase

The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free tyrosine. From Lineweaver-Burk plots and on the basis of a value of 0.83 for delta epsilon mM at 289 nm/iodine atom incorporated, the rate constant for transfer of an assumed enzyme-bound iodinium cation to thyroglobulin was estimated to be 6.7 X 10(7) and 2.3 X 10(7) M-1 s-1 in native (iodine content = 1.0%) and more iodinated (iodine content = 1.2%) thyroglobulins, respectively. This iodine-transferring reaction was stimulated by iodothyronines, similarly as observed in the reaction with free tyrosine. The iodination of thyroglobulin was inhibited by GSH, the inhibition being competitive with thyroglobulin. Thyroglobulin was oxidized in the presence of a thyroid peroxidase system without giving any appreciable change in absorbance around 300 nm. From stopped flow data, the oxidation was concluded to occur by way of two-electron transfer and the rate constant for the reaction of thyroid peroxidase Compound I with thyroglobulin was estimated to be 1.0 X 10(7) M-1 s-1. The stopped flow kinetic pattern was similar to that observed on the reaction with free tyrosine and monoiodotyrosine. About 6 mol of hydrogen peroxide were consumed per mol of thyroglobulin. Thyroid peroxidase catalyzed thyroglobulin-mediated oxidation of GSH, but lactoperoxidase did not.     

Nitrite-assisted peptide iodination and conjugation.

In this study, a simple method for selective iodination of peptides and proteins is established. Using angiotensin II as the model system, we demonstrate that nitrite catalyzed the selective iodination of the peptide at the N-terminus in an acidic solution. The N-terminal-iodinated peptides alkylated thiol-containing molecules such as N-acetylcysteine and glutathione to form peptide conjugates in a basic solution. Reactive species formed by increasing the pH of the reaction mixture of sodium nitrite and sodium iodide from 4 to 8 selectively iodinated peptides and proteins at tyrosine and histidine residues. These results show that nitrite is a useful catalyst for peptide and protein ligation.     

The effect of iodination of particular tyrosine residues on the hormonal activity of insulin

Insulin dissolved in aqueous or methanolic buffer was iodinated to give preparations containing an average of between one and five iodine atoms per insulin monomer. The resultant preparations were fragmented in various ways and the ratio of tyrosine to monoiodotyrosine and di-iodotyrosine was determined in each fragment. This has allowed the distribution of iodine between the combined A-chain tyrosine residues and the individual B-chain tyrosine residues to be determined. The hormonal activity of each of these iodinated insulin preparations was measured from their effect on the production of (14)CO(2) from [1-(14)C]glucose by isolated adipose cells. The results were interpreted as meaning that the iodination of tyrosine residue A19 or B16 leads to the inactivation of insulin. Speculations are made about the nature of an interaction between insulin and a receptor site on the target tissue.     

Iodination of a tyrosyl residue in staphylococcal alpha-toxin.

Iodination of staphylococcal alpha-toxin by the lactoperoxidase method resulted in the maximal incorporation of about 2.5 atoms of iodine per molecule of alpha-toxin. The iodination primarily involved a single tyrosine residue as shown by analysis of both cyanogen bromide and tryptic peptides. Iodination at a level of 1.2 iodine atoms per alpha-toxin molecule led to a dramatic decrease in the hemolytic and lethal activities, although no decrease in the binding of iodinated toxin to rabbit erythrocytes was observed (Cassidy and Harshman (1976), Biochemistry, the following paper in this issue). Monoiodinated alpha-toxin was found to have 15% of the specific hemolytic activity of native alpha-toxin. Incubation of rabbit erythrocytes with iodinated alpha-toxin led to a significant protection from the hemolytic activity of native alpha-toxin added later. The results show the modification of a single unique tyrosyl residue in alpha-toxin permits the resolution of alpha-toxin's biological activities from its cell binding activity.     

The sites of the lactoperoxidase-catalyzed iodination of human fibrinogen.

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.     

Iodination of Ricin D

Approximately five tyrosine residues of ricin D were iodinated preferentially under appropriate conditions probably forming diiodotyrosine. Iodination of this toxin carried out in 0.1 m phosphate buffer at pH 7.0 and 0°C for 60 min with a 20 fold molar excess of iodine per mole of protein, yielded a main component which appeared as a single band on polyacrylamide gel disc electrophoresis. Analysis of protein-bound radioactivity and the content of diiodotyrosine of ¹⁸¹I-labeled ricin D revealed that two tyrosine residues in the isoleucyl chain and three in the alanyl chain were substituted. The toxicity of iodinated ricin D decreased to one hundredth of that of native protein, However, the hemagglutinating activity of this protein was not affected by the iodination reaction.     

INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN

In the presence of iodine at pH 5.0–6.0 a solution of pepsin absorbs iodine and the specific proteolytic activity of the solution decreases. The activity is less than 1 per cent of the original activity when the number of iodine atoms per mol of pepsin is 35–40. If the pH is 4.5 or less, iodine reacts very slowly and there is a correspondingly slower loss in activity. Glycyl tyrosine reacts with iodine in a manner similar to pepsin. Experiments were performed to determine the extent to which oxidation of pepsin by iodine occurs during iodination, and if such oxidation were responsible for the loss in enzymatic activity. Although the results were not absolutely decisive, there seems to be no appreciable oxidation taking place during iodination and no relationship between the slight oxidation and loss in peptic activity. From a dialyzed preparation of completely iodinated pepsin which was inactive and contained 13.4 per cent bound iodine, 82 per cent of the iodine was obtained in a solution which analyzed as a solution of diiodo-tyrosine. Because of the presence of a material which contained no iodine and prevented quantitative crystallization, only 53 per cent of the iodine containing substance could be crystallized. This 53 per cent was, however, identified as diiodo-tyrosine. The part of the titration curve which in pepsin and most proteins represents the phenolic group of tyrosine was, in the curve for iodinated pepsin, shifted toward the acid region as expected. From these results, it appears that the loss in proteolytic activity of pepsin, when treated with iodine under the specified conditions, is due to the reaction of the iodine with the tyrosine in pepsin.     

Effect of N-bromosuccinimide-modification of tyrosine side chains of cardiotoxin II of the Indian cobra on biological activity

The essential role of tyrosine residue(s) of cardiotoxin II in the biological activity of the toxin was evaluated using N-bromosuccinimide. N-bromosuccinimide effected oxidation of the tyrosine residues in cardiotoxin II with enhancement in absorbance at 260 nm. The influence of various solvent media such as acetate-formate buffer (pH 4.0), 0.01 N H₂SO₄ (pH 2.0) and Tris-HCl buffer (pH 8.5) on oxidation of tyrosine residues was exa mined. In comparison with 0.01 N H₂S O₄, acetate-formate buffer could prevent secondary oxidations as revealed by lower consumption of oxidant, N-bromosuccinimide, to achieve oxidation. In Tris-HCl buffer oxidation of tyrosine did not take place effectively. N-iodo-succinimide caused only limited oxidation as evident from minor increase in absorbance at 260 nm. N-chlorosuccinimide was completely ineffective. Oxidation of cardiotoxin II with 3.75 equivalents of N-bromosuccinimide tyrosine residue led to complete loss of lethal activity. However, the derivative retained the ability to protect bacterial protoplasts from lysis in solutions of low tonicity. Unlike cardiotoxin II oxidized with N-chlorosuccinimide (50 equivalents/mol of toxin) which retained lethal activity as well as the ability to protect protoplasts from lysis, performic acid-oxidized toxin had lost both the activities.     

Iodination associated inactivation of beta-melanocyte stimulating hormone.

Contrary to other published reports, iodinated β-melanocyte stimulating hormone (β-MSH) is without biological activity as measured by frog skin bioassay, melanoma (mouse S-91) adenylate cyclase assay, or melanoma tyrosinase assay. Inactivation results in part from oxidation of the methionine residue by the chloramine T and sodium metabisulfite used in the iodination reaction. Replacement of the methionine of β-MSH with norleucine by solid phase synthesis results in an analogue which is more resistant, but not completely resistant, to inactivation. Thus, in order to obtain a biologically active radioligand for radioreceptor studies, further tailoring of the hormone and/or modification of the iodination procedure will be needed.     

Targeted tyrosine iodination in a multi-tyrosine vasopressin analog.

Iodination of the conserved 2-tyrosine (Tyr(2)) residue in the pressin and tocin rings of arginine- or lysine-vasopressin (AVP or LVP), and oxytocin, respectively, impairs binding to their respective receptors. Synthetic antagonists that have their Tyr(2) either replaced by another amino acid or irreversibly blocked by an O-methyl or O-ethyl ether, but have, instead, an iodinatable phenol moiety outside the pressin/tocin ring, are used for radiolabeling. We explored another approach to avoid iodinating Tyr(2) by capping this residue with a reversible O-acetyl group, incorporated during peptide synthesis. The O-acetyl-Tyr(2) LVP peptide, with a free iodinatable tyrosine attached to the epsilon-amine of 8-lysine, is iodinated at a neutral pH and purified by reverse-phase high-pressure liquid chromatography (HPLC) at an acidic pH, conditions under which the O-acetyl groups are stable. Deacetylation with hydroxylamine is selective, and leaves intact the disulfide bridge. The marked shortening of the HPLC retention time after deblocking produces a chemically homogeneous label, iodinated exclusively on the free tyrosine residue attached to the epsilon-amine of LVP. Hitherto, this (125)I labeled vasopressin agonist could be obtained only in low yield, via conjugation labeling with iodinated N-t-Boc-tyrosine succinimidyl ester. This fully reversible tyrosine protection strategy does not require special equipment, and retains the conserved Tyr(2), typical of vasopressin and oxytocin agonists.     

Identification of a crucial amino acid responsible for the loss of specifying FGFR1–KLB affinity of the iodinated FGF21

Previous studies suggest that specific binding to the complex consisting of fibroblast growth factor receptor-1 (FGFR1) and the coreceptor beta-Klotho (KLB) is the premise for human FGF19 and FGF21 activating the downstream signaling cascades, and regulating the metabolic homeostasis. However, it was found that human FGF21 loses its ability to bind to FGFR1–KLB after iodination with Na¹²⁵I and chloramine T, whereas human FGF19 retained its affinity for FGFR1–KLB even after iodination. The molecular mechanisms underlying these differences remained elusive. In this study, we first demonstrated that an intramolecular disulfide bond was formed between cysteine-102 and cysteine-121 in FGF21, implying that the oxidation of the cysteine to cysteic acid, which may interfere with the active conformation of FGF21, did not occur during the iodination procedures, and thus ruled out the possibility of the two conserved cysteine residues mediating the loss of FGF21 binding affinity to FGFR1–KLB upon iodination. Site-directed mutagenesis and molecular modeling were further applied to determine the residue(s) responsible for the loss of FGFR1–KLB affinity. The results showed that mutation of a single tyrosine-207, but not the other five tyrosine residues in FGF21, to a phenylalanine retained the FGFR1–KLB affinity of FGF21 even after iodination, whereas replacing the corresponding phenylalanine residue with tyrosine in FGF19 did not alter its binding affinity to FGFR1–KLB, but decreased the receptor binding ability of the iodinated protein, suggesting that tyrosine-207 is the crucial amino acid responsible for the loss of specifying FGFR1–KLB affinity of the iodinated FGF21.     

Site-directed mutagenesis and molecular modeling identify a crucial amino acid in specifying the heparin affinity of FGF-1.

Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with Na(125)I and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl(2). Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.     

Efficiency of methemoglobin, hemin and ferric citrate in catalyzing protein tyrosine nitration, protein oxidation and lipid peroxidation in a bovine serum albumin-liposome system: Influence of pH

Protein tyrosine nitration, protein oxidation and lipid peroxidation are nitrative/oxidative modification of protein and lipids. In this paper, a BSA (bovine serum albumin)-lecithin liposome system was used to study the nature of different forms of iron, including methemoglobin, hemin and ferric citrate, in catalyzing H₂O₂-nitrite system to oxidize protein and lipid as well as nitrate protein. It was found that in pH range of 5.0-9.0, in pure BSA solution or pure liposome solution, hemin and methemoglobin catalyzed protein tyrosine nitration and lipid peroxidation were decreased with the increasing of pH, while hemin and methemoglobin catalyzed protein oxidation was significantly and moderately increased, respectively. Lipid completely inhibited hemin catalyzed protein tyrosine nitration but only partially inhibited methemoglobin catalyzed protein tyrosine nitration, and its inhibitory effect on hemin induced protein oxidation was also more pronounced. In addition, BSA showed more efficient in inhibiting hemin and ferric citrate induced lipid peroxidation. At the same condition, ferric citrate was relatively ineffective in all tests. Considering protein tyrosine nitration, protein oxidation and lipid oxidation as overall oxidative damage, these results indicated that methemoglobin is more toxic than hemin and ferric citrate, the degradation procedure of heme containing macromolecules, e.g. hemoglobin to hemin and finally to low molecular weight bounded iron, is step by step detoxification. These results provide fundamental knowledge on oxidative/nitrative of biomolecules in lipid-protein coexistence system.     

Free diiodotyrosine effects on protein iodination and thyroid hormone synthesis catalyzed by thyroid peroxidase.

Free diiosotyrosine exerts two opposite effects on the reactions catalyzed by thyroid peroxidase, thyroglobulin iodination and thyroid hormone formation. 1. Inhibition of thyroglobulin iodination catalyzed by thyroid peroxidase was observed when free diiodotyrosine concentration was higher than 5 muM. This inhibition was competitive, suggesting that free diiodotyrosine interacts with the substrate site(s) of thyroid peroxidase. Free diiodotyrosine also competively inhibited iodide peroxidation to I2. 2. Free diiodotyrosine, when incubated with thyroid peroxidase in the absence of iodide was recovered unmodified; in the presence of iodide an exchange reaction was observed between the iodine atoms present in the diiodotyrosine molecule and iodide present in the medium. Using 14C-labelled diiodotyrosine, 14C-labelled non-iodinated products were also observed, showing that deiodination occurred as a minor degradation pathway. However, no monoiodo[14C]tyrosine or E114C]tyrosine were observed. Exchange reaction between free diiototyrosine and iodide is therefore direct and does not imply deiodination-iodination intermediary steps. Thyroglobulin inhibits diiodotyrosine-iodide exchange and vice versa, again suggesting competition for both reactions. These results support, by a different experimental approach, the two-site model for peroxidase previously described by us in this journal. 3. Free diiodotyrosine when present at a very low concentration, 0.05 muM, exerts a stimulatory effect on throid hormones synthesis. The relationship between diiodotyrosine concentration and thyroid hormone synthesis give an S-shaped curve, suggesting that free diiodotyrosine acts as a regulatory ligand for thyroid peroxidase. Evidence is also presented that free diiodotyrosine is not incorporated into thyroid hormones. Therefore, thyroid peroxidase catalyzes only intra-molecular coupling between iodotyrosine hormonogenic residues. 4. Finally, although no direct proof exists that these free diiodotyrosine effects upon thyroglobulin iodination and thyroid hormone synthesis are physiologically significant, such a possibility deserves further investigation.