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.     

Chloramine-T in radiolabeling techniques. III. Radioiodination of biomolecules containing thioether groups

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.     

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.     

Studies on the mechanism of the iodination of tyrosine by lactoperoxidase

Studies with lactoperoxidase showed that a highly reactive intermediate is produced (on the enzyme) from I- and H2O2 which then diffuses from the enzyme and very rapidly and indiscriminately iodinates any Tyr or peptides containing Tyr which are in the same solution. The evidence supporting these conclusions follows. 1) The rate followed the Michaelis-Menten pattern with I- and H2O2 while the concentration of Tyr peptides had no measurable effect on the rate; 2) the rates of reaction were independent of the type of peptide in which Tyr was located; 3) the amount of iodination which had occurred after the reaction had gone to completion and the amounts of monoiodination and diiodination after completion of the reaction were independent of the peptide type, the pH, the solvent polarity, or the ionic strength; 4) competition for reaction by two very different Tyr peptides depended only on their initial concentrations; and 5) iodination of a large protein occurred through a dialysis membrane. Free Tyr was iodinated at the same rate as Tyr peptides by lactoperoxidase, but monoiodotyrosine and m-fluorotyrosine were iodinated at one-half that rate. The results also showed that one can choose ratios of [peptide] to [H2O2] such that monoiodination is maximized relative to diiodination. It was also found that the iodination capacity of a mixture of I- and H2O2 with lactoperoxidase (when Tyr was absent) was only slowly dissipated. Finally, the results showed that lactoperoxidase can be used to brominate and chlorinate Tyr peptides at a slow rate.     

Myeloperoxidase-catalyzed iodination and coupling.

Myeloperoxidase (MPO), which displays considerable amino acid sequence homology with thyroid peroxidase (TPO) and lactoperoxidase (LPO), was tested for its ability to catalyze iodination of thyroglobulin and coupling of two diiodotyrosyl residues within thyroglobulin to form thyroxine. After 1 min of incubation in a system containing goiter thyroglobulin, I-, and H2O2, the pH optimum of MPO-catalyzed iodination was markedly acidic (approximately 4.0), compared to LPO (approximately 5.4) and TPO (approximately 6.6). The presence of 0.1 N Cl- or Br- shifted the pH optimum for MPO to about 5.4 but had little or no effect on TPO- or LPO-catalyzed iodination. At pH 5.4, 0.1 N Cl- and 0.1 N Br- had a marked stimulatory effect on MPO-catalyzed iodination. At pH 4.0, however, iodinating activity of MPO was almost completely inhibited by 0.1 N Cl- or Br-. Inhibition of chlorinating activity of MPO by Cl- at pH 4.0 has been previously described. When iodination of goiter thyroglobulin was performed with MPO plus the H2O2 generating system, glucose-glucose oxidase, at pH 7.0, the iodinating activity was markedly increased by 0.1 N Cl-. Under these conditions iodination and thyroxine formation were comparable to values observed with TPO. MPO and TPO were also compared for coupling activity in a system that measures coupling of diiodotyrosyl residues in thyroglobulin in the absence of iodination. MPO displayed very significant coupling activity, and, like TPO, this activity was stimulated by a low concentration of free diiodotyrosine (1 microM). The thioureylene drugs, propylthiouracil and methimazole, inhibited MPO-catalyzed iodination both reversibly and irreversibly, in a manner similar to that previously described for TPO-catalyzed iodination.     

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.     

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.     

The effect of adjacent residues on the reactivity of tyrosyl with iodine

The effect of the adjacent amino acid side chain groups on the iodination rate of the tyrosine was studied. The model peptides used were Gly-Tyr-Gly, Leu-Tyr-Leu, Glu-Tyr-Glu, and Lys-Tyr-Lys, in which the tyrosine is sandwiched between two hydrophobic, two negatively charged, or two positively charged residues. The results show only minor differences in the iodination rate of tyrosine in these four peptides. These differences are very small in comparison with those previously observed between the tyrosines of kappa Bence-Jones proteins.     

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.     

Relation between the iodination of human thyroglobulin and the cleavage of the hormone peptide 26K N-terminal

At moderate iodination levels (about 20 iodine atoms/mol) human thyroglobulin yields after reduction and alkylation a hormone (T4)-containing N-terminal peptide of 26K. Further iodination of the thyroglobulin in vitro results in the cleavage of this part of the molecule into smaller peptides of 22K and 18K. A precursor-product relationship between the 26K peptide segment and the latter was established by showing an identical N-terminal T4-containing sequence in the 3 peptides. Cleavage of peptide bonds in the 26K segment to give the smaller fragments could possibly be related to the formation of another hormone residue.     

A versatile procedure for the radioionization of proteins and labeling reagents

For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.     

A versatile procedure for the radioiodination of proteins and labeling reagents

For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.     

Relations of iodination, hormonosynthesis and proteolytic cleavage in human thyroglobulin

Normally iodinated thyroglobulin (Tg) contains low molecular weight hormone-rich peptides associated to the bulk of the molecule by disulfide bridges. It is shown, with the assistance of in vitro iodination experiments using different iodine concentrations and various incubation times, that the proteolytic cleavage giving rise to the 26 K hormonopeptide in human Tg is part of a coupling reaction rather than iodination. This cleavage may be a preliminary event related to a facilitation in the release of thyroid hormones.     

Chemical Modification of Neutral Protease from Bacillus subtilis var. amylosacchariticus: Assignment of Tyrosyl Residues Iodinated

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0°C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153—159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.     

Effects of iodination on the distribution of peptide hormones in aqueous two-phase polymer systems

1. The effect of iodination on the distribution of peptide hormones into the aqueous two-phase dextran-polyethylene glycol system and on the solubility of these hormones in aqueous polyethylene glycol and in water was assessed. Hormones that were studied included insulin, glucagon and parathyroid hormone. 2. The partition coefficient of native insulin in the dextran-polyethylene glycol system showed a minimum (about 1) near the isoelectric point of the hormone (pH 5). Partial iodination of insulin (one atom per molecule) caused little change in the distribution of the hormone. More extensive iodination markedly decreased the partition coefficient in the region of the isoelectric point and displaced the pH value at which the partition coefficient was a minimum towards lower values. 3. The solubility of native insulin in aqueous polyethylene glycol and in water showed a pH-dependence similar to that observed for the distribution in the dextran-polyethylene glycol system. Iodination of insulin decreased the solubility of the hormone in polyethylene glycol and in water in parallel, and decreased the pH value at which solubility was a minimum. The changes in solubility correlated with the degree of iodination and accounted for the changes in distribution observed at high concentrations of insulin. 4. Comparable effects of iodination on distribution and solubility were also observed with glucagon. 5. At concentrations of insulin below its maximum solubility, serum proteins caused a decrease in the partition coefficient of iodinated hormone, but not of native hormone. These effects correlated with the degree of iodination and resulted from a co-precipitation of iodinated insulin with serum proteins.     

Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus.

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.     

The effect of lactoperoxidase-catalyzed iodination on the integrity of mitochondrial membranes.

The effect of lactoperoxidase-catalyzed iodination on rat liver mitochondria was investigated. A change from the condensed to the swollen conformation is observed by electron microscopy after extensive iodination of the mitochondria. The outer membrane breaks after incorporation of 0.2 nmol or more iodine atoms per mg of mitochondrial protein releasing adenylate kinase, a soluble enzyme located in the intermembrane space. Further iodination of the mitochondria ruptures the inner membrane, releasing proteins such as glutamic dehydrogenase from the matrix space. Lipid peroxides and I₂ are not intermediates in the disruptive effect of extensive lactoperoxidase-catalyzed iodination on the membranes. During iodination at pH 6.5 almost no release of protein or glutamic dehydrogenase activity is detectable and the loss of adenylate kinase activity from the particulate is diminished. The effect of extensive iodination on mitochondrial membranes limits the amount of iodide which can be incorporated with the lactoperoxidase membrane-labeling procedure when this technique is used as a surface probe of mitochondrial membranes.     

Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodine and Isolation of Peptide by One Step Ether Precipitation

The S-acetamidomethyl (Acm) protecting group is widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report a simple method for simultaneous post-cysteine (Acm) group removal quenching of iodination and isolation. Use of large volumes of diethyl ether for direct precipitation action of the oxidized peptide from the 90 or 95% aqueous acetic acid solution affords nearly quantitative recovery of largely iodine-free peptide ready for direct purification. It was successfully applied to the synthesis of various peptides including human insulin-like peptide 3 analogues. Although recovery yields were comparable to the traditionally used ascorbic acid quenching method, this new approach offers significant advantages such as more simple utility, minimal side reactions, and greater cost effectiveness.     

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.