Effects of Antioxidants on the Hydrogen Peroxide-Mediated Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor and Human Parathyroid Hormone Fragment 13-34

No HeadingPurpose. The effects and mechanisms of different antioxidants, methionine, glutathione, acetylcysteine, and ascorbic acid (AscH₂), on the oxidation of methionine residues in granulocyte colony-stimulating factor (G-CSF) and human parathyroid hormone fragment 13-34 (hPTH 13-34) by hydrogen peroxide (H₂O₂) were quantified and analyzed.Methods. The rates of oxidation of methionine residues in G-CSF were determined by peptide mapping analyses, and the oxidation of methionine residue in hPTH 13-34 was quantified by reverse-phase HPLC.Results. At pH 4.5, free methionine reduces, glutathione and acetylcysteine have no obvious effect on, and AscH₂ promotes the rates of oxidation of methionine residues in G-CSF. The H₂O₂-induced oxidation rate constants for free methionine, acetylcysteine, and glutathione at pH 4.5 were measured to be 32.07, 1.00, and 1.63 M^-1h^-1, respectively, while the oxidation rate constant for Met¹, the most readily oxidizable methionine residue in G-CSF, is 13.95 M^–1h^–1. Therefore, the different effects of free methionine, acetylcysteine, and glutathione on the rates of oxidation of methionine residues in G-CSF are consistent with their different reactivity toward oxidation by H₂O₂. By using hPTH 13-34, the effect of AscH₂ on the H₂O₂-induced oxidation of methionine residue was quantified, and the mechanisms involved were proposed. Because of the presence of trace transition metal ions in solution, at low concentrations, AscH₂ is prone to be a prooxidant, increasing the hydroxyl radical (OH) production rate via Fenton-type reactions. In addition to peroxide oxidation, these radicals lead to the degradation of hPTH 13-34 to smaller peptide fragments. At high concentrations, AscH₂ tends to act as an OH scavenger. EDTA inhibits OH production and thus eliminates the degradation of hPTH 13-34 by forming complexes with transition metal ions. However, the rate of oxidation of the methionine residue in hPTH 13-34 increases as the concentration of AscH₂ is increased from 0 to 200 mM, and the reason for this is still not clear.Conclusions. Our results demonstrate that free methionine is an effective antioxidant to protect G-CSF against methionine oxidation at pH 4.5. Acetylcysteine and glutathione are not effective antioxidants at pH 4.5. Their oxidation rates at different pH values imply that they would be much more effective antioxidants than free methionine at alkaline conditions. AscH₂ is a powerful electron donor. It acts as a prooxidant in the conditions in this study and is unlikely to prevent oxidation by H₂O₂ in protein formulation, whether or not EDTA is present.     

Oxidation of recombinant methionyl human granulocyte colony stimulating factor

The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of this system was confirmed with micellar electrokinetic capillary chromatography. In the pH range 1.9–7.5, the k_obs value for the oxidation process is constant. Above pH 7.5, k_obs tends to increase with increasing pH. In the pH range 1.9–11.8, four oxidation products were detected in RP-HPLC. Mass spectrometric analysis revealed that one mono-, one di- and two trioxidation products were formed. Using the cyanogen bromide cleavage method the nature of the oxidation products was determined. The mono-oxidation product is the protein with Met¹²¹ oxidized, while the dioxidation product has oxidized Met¹²¹ and Met¹²⁶ residues. The trioxidation products are the proteins with Met¹²¹, Met¹²⁶ and Met¹³⁷ or Met⁰, Met¹²¹ and Met¹²⁶ oxidized.     

Oxidized Recombinant Human Growth Hormone That Maintains Conformational Integrity

Chemical degradations often induce changes in protein conformation and thus influence protein activity and protein stability in solutions. One difficulty in studying of chemical degradations on protein aqueous properties is to obtain sufficient amount of chemically degraded protein which is well characterized. Chemical degradation protocols that are often used may induce also conformation changes and aggregation of the protein. In this article we studied the effect of methionine oxidation on the conformation of recombinant human growth hormone (r-hGH). In literature it is reported that oxidation of methionine residues induces conformation changes on r-hGH. In our study, oxidation of r-hGH was performed by incubation with hydrogen peroxide under mild conditions. Mass spectrometry and chromatographic analysis revealed that oxidation with hydrogen peroxide resulted in more than 90% of oxidized r-hGH. By extensive spectroscopic characterizations no detectable change in conformation and aggregation of r-hGH after oxidation was found. In conclusion, mild oxidation conditions led to selective oxidation of the two more accessible methionine residues of r-hGH (Met¹⁴ and Met¹²⁵) and did not results in any conformation change of the protein. These findings prove that oxidation of human growth hormone does not influence protein conformation and demonstrate the importance of employing mild conditions during production of oxidized protein.     

Protection of methionine in peptides during iodination by sulfonium salt formation

A method for the prevention of methionine oxidation during iodination of tyrosine containing peptides is reported. The methionine containing peptide is converted into the corresponding S-tert.-butylsulfonium derivative, which is iodinated using iodine monochloride. After removal of the S-tert.-butyl group and purification, sulfoxide-free 3,5 diiodotyrosine (Dit) peptides were obtained. Dit⁸-substance P, Dit⁸-physalaemin^6–11 and Dit¹, Met⁵-enkephalin were synthesized by this route. Tritium labeling of Dit¹, Met⁵-enkephalin yielded ³H-enkephalin with a specific radioactivity of 38 Ci/mmol.     

Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ～200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.     

In Vitro Methionine Oxidation of Recombinant Human Leptin

Purpose. To investigate the role and importance of the four methionines in recombinant human leptin, and the effect of methionine oxidation in leptin structural stability and biological activity. Methods. Oxidized leptin derivatives were prepared in the presence of H₂O₂ and t-butylhydroperoxide, separated by RP-HPLC, and characterized by peptide mapping and LC/MS. Their biophysical and biological properties were studied. Results. Six major species of oxidized leptins were detected: two mono-oxidized, one di-oxidized, two tri-oxidized, and one tetra-oxidized. Further oxidation at cystine disulfide was also detected. Kinetic analysis indicated that oxidation at Met¹ and Met⁶⁹ proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k ₁ and k ₆₉, were 1.5 × 10^–3 and 2.3 × 10^–4 min^–1. No change in the secondary or tertiary structure was detected for Met¹ mono-oxidized and Met¹, Met⁶⁹ di-oxidized leptins. The Met¹ mono-oxidized leptin retained full potency as compared to native leptin. A slight decrease of thermostability and a significant loss of the in vitro bioactivity were observed for Met¹, Met⁶⁹ di-oxidized leptin. Both Met⁵⁵ and Met¹³⁷ were not oxidized in t-butylhydroperoxide but only in H₂O₂. They appeared to be much less accessible to oxidation and might interact with the hydrophobic core structure of the leptin molecule. Conclusions. The oxidation of leptin occurred in the order of Met¹ > Met⁶⁹ >> Met⁵⁵ Met¹³⁷, and the importance for maintaining leptin structural integrity was Met⁵⁵ Met¹³⁷ >> Met⁶⁹ Met¹. Met⁶⁹, but not Met¹, plays a critical role in the protein stability and activity.     

Chemical Pathways of Peptide Degradation. V. Ascorbic Acid Promotes Rather than Inhibits the Oxidation of Methionine to Methionine Sulfoxide in Small Model Peptides

The effect of primary structure and external conditions on the oxidation of methionine to methionine sulfoxide by the ascorbate/Fe³⁺ system was studied in small model peptides. Degradation kinetics and yield of sulfoxide formation were dependent on the concentration of ascorbate and H⁺, with a maximum rate observed at pH 6–7. Phosphate buffer significantly accelerated the peptide degradation compared to Tris, HEPES, and MOPS buffers; however, the formation of sulfoxide was low. The oxidation could not be inhibited by the addition of EDTA. Other side products besides sulfoxide were observed, indicating the existence of various other pathways. The influence of methionine location at the C terminus, at the N terminus, and in the middle of the sequence was investigated. The presence of histidine in the sequence markedly increased the degradation rate as well as the sulfoxide production. The histidine catalysis of methionine oxidation occurred intramolecularly with a maximum enhancement of the oxidation rate and sulfoxide production when one residue was placed between the histidine and the methionine residue.     

Radioiodinated [D-Ala2, Met 5] enkephalin

¹²⁵I[D-Ala², Met⁵] enkephalin with high specific activity (122–185 Ci/mmol) was prepared and purified by Sep-Pak C₁₈ reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved ¹²⁵I[D-Ala², Met⁵] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same R_f values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24d?. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing ¹²⁵I[D-Ala², Met⁵] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala², Met⁵] enkephalin and [D-Ala², Leu⁵] enkephalin for 50% inhibition of ¹²⁵I[D-Ala², Met⁵] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the ¹²⁵I[D-Ala², Met⁵] enkephalin binding to any significant extent. The ¹²⁵I[d-Ala², Met⁵] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Mitigation of Oxidation in Therapeutic Antibody Formulations: a Biochemical Efficacy and Safety Evaluation of <Emphasis Type="Italic">N</Emphasis>-Acetyl-Tryptophan and L-Methionine

Purpose

Biotherapeutics can be susceptible to oxidation during manufacturing and storage. Free L-methionine is known to protect methionine residues in proteins from oxidation. Similarly, free tryptophan and other indole derivatives have been shown to protect tryptophan residues from oxidation. N-acetyl-DL-tryptophan was previously identified as a potentially superior antioxidant to tryptophan as it has a lower oxidation potential and produces less peroxide upon light exposure. This study sought to confirm the antioxidant efficacy and safety of N-acetyl-DL-tryptophan and L-methionine as formulation components for biotherapeutic drugs.

Methods

Antibodies were subjected to AAPH and light exposure in the presence of N-acetyl-DL-tryptophan and L-methionine. Oxidation in relevant CDR and Fc residues was quantified by peptide map. In silico, in vitro, and in vivo studies were performed to evaluate the safety of N-acetyl-DL-tryptophan and L-methionine.

Results

Peptide mapping demonstrated that N-acetyl-DL-tryptophan was effective at protecting tryptophans from AAPH stress, and that the combination of N-acetyl-DL-tryptophan and L-methionine protected both tryptophan and methionine from AAPH stress. The safety assessment suggested an acceptable safety profile for both excipients.

Conclusions

N-acetyl-tryptophan and L-methionine effectively reduce the oxidation of susceptible tryptophan and methionine residues in antibodies and are safe for use in parenteral biotherapeutic formulations.

     

The Kinetics of Relaxin Oxidation by Hydrogen Peroxide

In this study, hydrogen peroxide was used to study the oxidation of rhRlx under various conditions. Oxidation of rhRlx occurred at both of the two methionines on the B chain, Met B(4) and Met B(25), as expected from the three-dimensional structure of the molecule, which shows that these two residues are located on the surface of the molecule and exposed to solvent. The reaction produced three different oxidized forms of rhRlx containing either Met B(4) sulfoxide, Met B(25) sulfoxide, or both residues oxidized. The corresponding sulfone was not formed under these conditions. The oxidation at the two methionines proceeded independently from each other but Met B(25) was oxidized at a significantly faster rate than Met B(4). The fact that the rate of oxidation at Met B(25) was identical to the rate of oxidation of free methionine and that of two model peptides mimicking the residues around Met B(4) and Met B(25) suggests that the lower reactivity at Met B(4) was due to steric hindrance, and at least in this case, neighboring groups do not influence the oxidation kinetics of methionine residues. The reaction was independent of pH, ionic strength, and buffer concentration in the range studied. The enthalpy of activation for the reaction was approximately 10–14 kcal mol^–1, with an entropy of activation of the order of –30 cal K^–1 mol^–1. These data are consistent with previously published mechanisms for organic sulfide oxidation by alkyl hydroperoxides.     

Comparison Between Light Induced and Chemically Induced Oxidation of rhVEGF

Purpose. The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. Methods. A liquid formulation of rhVEGF was exposed to fluorescent light (2 × 10⁴ lux for up to 4 weeks), hydrogen peroxide (H₂O₂), or t-butylhydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. Results. Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78,81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%, possibly due to the role of Met18 in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H₂O₂, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H₂O₂ and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. Conclusions. Methionine residues with exposed surface areas greater than 65 Ų and sulfur surface areas greater than 16 Ų were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H₂O₂ to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 Ų, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.     

A comprehensive picture of non-site specific oxidation of methionine residues by peroxides in protein pharmaceuticals

In this article, a comprehensive picture of the oxidation of protein pharmaceuticals by peroxides is developed based on our earlier computational and experimental studies. We propose a new mechanism, the water-mediated mechanism, for the oxidation of methionine residues, and it has been shown to satisfy all available experimental data including new data reported here. Based on the water-mediated mechanism, we found a structural property, average 2-shell water coordination number, that correlates well to the relative rates of oxidation of methionine groups. We used this to study the oxidation of granulocyte colony-stimulating factor (G-CSF) and 1-34 human parathyroid hormone hPTH(1-34). We believe that this comprehensive picture should aid researchers in the pharmaceutical sciences to develop solvent formulations for therapeutic proteins in a more rational way.     

Design,Synthesis, and Biological Evaluation of Novel Peptide Gly3‐MC62 Analogues as Potential Antidiabetic Agents

Two series of conformationally constrained analogues from Gly ³ ‐ MC 62 were designed by scanning the residues Lys¹, Thr², Met⁴, Lys⁵, Met^7, and Ala⁸ with an i‐(i + 2) lactam bridge consisting of a Glutamic acid–xaa–lysine (Glu–Xaa–Lys) scaffold and a diproline fragment. They were synthesized and evaluated for their antihyperglycemic effects. Through screening in normal and mice with diabetes mellitus, peptides II ‐5 , III ‐3 , III ‐4 , and III ‐5 showed significant improvement in antihyperglycemic and antioxidative activities compared with Gly ³ ‐ MC 62 , especially the compound III ‐4 . The primary mechanism of the compounds ( II ‐5 , III ‐3 , III ‐4 , and III ‐5 ) underlying this effect is the islet β‐cells against oxidative damage induced by STZ, and III ‐4 ‐treated mice showed considerable improvement in the preservation of beta cells in the pancreatic islets of DM mice. These data suggested that III ‐4 could be candidate for the future treatment of diabetes mellitus.     

Oxidation of Human Insulin-like Growth Factor I in Formulation Studies: Kinetics of Methionine Oxidation in Aqueous Solution and in Solid State

Purpose. The aim of this work was to study the kinetics of oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I)¹ in aqueous solution and in the solid state by the aid of quantification of oxygen. Methods. The oxidized form of hIGF-I was characterized by tryptic peptide analysis, RP-HPLC and FAB-MS and quantified by RP-HPLC. The oxygen content was quantified polarographically by a Clark-type electrode. Results. Second-order kinetics with respect to amount of protein and dissolved oxygen was found to be appropriate for the oxidation of methionine in hIGF-I. The rate constants ranged from 1 to 280 M^–1 month^–l and had an activation energy of 95 (+/–4) kJ/mole. Light exposure, storage temperature and oxygen content were found to have a considerable impact on the oxidation rates. No significant difference in reaction rates was found for the oxidation of hIGF-I in aqueous solution or in the solid state. A method for decreasing the oxygen content in aqueous solution without purging is described. Conclusions. Polarographic quantification of dissolved oxygen makes it possible to establish the kinetics for oxidation of proteins. The oxidation of methionine in hIGF-I appears to follow second-order kinetics.     

Triethylenetetramine prevents insulin aggregation and fragmentation during copper catalyzed oxidation

Metal catalyzed oxidation via the oxidative system Cu²⁺/ascorbate is known to induce aggregation of therapeutic proteins, resulting in enhanced immunogenicity. Hence, inclusion of antioxidants in protein formulations is of great interest. In this study, using recombinant human insulin (insulin) as a model, we investigated the ability of several excipients, in particular triethylenetetramine (TETA), reduced glutathione (GSH) and ethylenediamine tetraacetic acid (EDTA), for their ability to prevent protein oxidation, aggregation, and fragmentation. Insulin (1 mg/ml) was oxidized with 40 μM Cu²⁺ and 4 mM ascorbic acid in absence or presence of excipients. Among the excipients studied, 1 mM of TETA, EDTA, or GSH prevented insulin aggregation upon metal catalyzed oxidation (MCO) for 3 h at room temperature, based on size exclusion chromatography (SEC). At lower concentration (100 μM), for 72 h at +4 °C, TETA was the only one to inhibit almost completely oxidation-induced insulin aggregation, fragmentation, and structural changes, as indicated by SEC, nanoparticle tracking analysis, light obscuration particle counting, intrinsic/extrinsic fluorescence, circular dichroism, and chemical derivatization. In contrast, GSH had a slight pro-oxidant effect, as demonstrated by the higher percentage of aggregates and a more severe structural damage, whereas EDTA offered substantially less protection. TETA also protected a monoclonal IgG1 against MCO-induced aggregation, suggesting its general applicability. In conclusion, TETA is a potential candidate excipient for inclusion in formulations of oxidation-sensitive proteins.     

The effect of COMT Val158 Met genotype on decision-making and preliminary findings on its interaction with the 5-HTTLPR in healthy females

Poor decision-making is inherent to several psychiatric conditions for which a genetic basis may exist. We previously showed that healthy female volunteers homozygous for the short allele (s/s) of the serotonin transporter length polymorphic region (5-HTTLPR) chose more often cards from disadvantageous decks in the Iowa Gambling Task (IGT), which measures decision-making, than long (l) allele carriers. The 5-HTTLPR and catechol-O-methyltransferase (COMT) Val¹⁵⁸ Met polymorphism affect the same set of neuronal structures. Therefore, we explored the effect of the (COMT) Val¹⁵⁸ Met polymorphism on IGT performance and its interaction with the 5-HTTLPR in the same subjects in this study. We observed that subjects homozygous for methionine (Met/Met) chose more disadvantageously than subjects homozygous for valine (Val/Val). s/s-Met/Met-subjects appeared to show the poorest IGT performance of all possible combinations of 5-HTTLPR and COMT allelic variants. Using the Expectancy-Valence model, no differences were found for the three different 5-HTTLPR or COMT genotypes regarding (i) attention to wins versus losses, (ii) updating rate, or (iii) response consistency. However, subjects with at least one Met-allele were paying more attention to wins than subjects with no Met-alleles. We discuss whether a common neuronal mechanism relates to s- and Met-allele-related deficits in updating and/or processing of choice outcome to guide subsequent choices in this gamble-based test.     

Prediction of the Hydrogen Peroxide–Induced Methionine Oxidation Propensity in Monoclonal Antibodies

Methionine oxidation in therapeutic antibodies can impact the product's stability, clinical efficacy, and safety and hence it is desirable to address the methionine oxidation liability during antibody discovery and development phase. Although the current experimental approaches can identify the oxidation-labile methionine residues, their application is limited mostly to the development phase. We demonstrate an in silico method that can be used to predict oxidation-labile residues based solely on the antibody sequence and structure information. Since antibody sequence information is available in the discovery phase, the in silico method can be applied very early on to identify the oxidation-labile methionine residues and subsequently address the oxidation liability. We believe that the in silico method for methionine oxidation liability assessment can aid in antibody discovery and development phase to address the liability in a more rational way.     

CONFORMATIONAL ENERGY CALCULATIONS ON ENKEPHALINS AND ENKEPHALIN ANALOGS:

Conformational energy calculations were carried out on the peptide enkephalins (ENK) and selected analogs to find those conformers of low energy. The analogs studied include [D-Ala²]Enk-NH₂, [D-Ala²]Enk, [D-Met², Pro⁵]Enk-NH₂, [D-Ala², D-Phe⁵]Enk, [D-Ala², D-Leu⁵]Enk, [D-Ala², (N-Me)Phe⁴, Met⁵] Enk-NH₂ and [D-Ala², (N-Me)Met⁵]Enk-NH₂. When the low-energy conformers for all the analogs are compared, different allowed backbone conformations are found which orient the functional side-chains such that three classifications of structures appear. Each classification shows a unique configuration of side- chain positions in space even though different backbone conformations are found within each classification.     

Oxidation of Recombinant Human Interleukin-2 by Potassium Peroxodisulfate

Purpose. The oxidation of recombinant human interleukin-2 (rhIL-2) by potassium peroxodisulfate (KPS) with or without N,N,N,N-tetramethylethylenediamine (TEMED), which are used for the preparation of dextran-based hydrogels, was investigated. Methods. The oxidation of (derivatives of) methionine, tryptophan, histidine and tyrosine, as well as rhIL-2 was investigated. Both the oxidation kinetics (RP-HPLC) and the nature of the oxidation products (mass spectrometry) were studied as a function of the KPS and TEMED concentration, and the presence of a competitive antioxidant, methionine. Results. Under conditions relevant for the preparation of rhIL-2 loaded hydrogels, only methionine and tryptophan derivatives were susceptible to oxidation by KPS. The oxidation of these compounds was inhibited once TEMED was present, suggesting that the peroxodisulfate anion, rather than the radicals formed in the presence of TEMED, is the oxidative species. KPS only induced oxidation of the four methionines present in rhIL-2, whereas the tryptophan residue remained unaffected. The radicals, formed after KPS decomposition by TEMED, induced some dimerization of rhIL-2. The oxidation of rhIL-2 could be substantially reduced by the addition of methionine, or by pre-incubation of KPS with TEMED. Conclusions. Only the methionine residues in rhIL-2 are oxidized by KPS. The extent of oxidation can be minimized by a proper selection of the reaction conditions.     

Coupling difficulty following replacement of Tyr with HOTic during synthesis of an analog of an EGF B-loop fragment

During Fmoc synthesis of an analog, [Abu^20,31, HOTic²²]hEGF(20–31), of a fragment, Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys, of the B-loop of human EGF, conductivity measurements showed that increased time was necessary for coupling and complete deprotection of the residues Met²¹ and Abu²⁰ which followed the HOTic²². Use of different active ester-forming reagents, including HOBt and BOP, did not increase the yield. Use of symmetrical anhydride with extended coupling time increased the yield but did not complete the coupling. It appears that inclusion of HOTic in place of Tyr to introduce conformational constraint to peptide analogs can cause or augment a tendency towards conformations with increasing occlusion of N-terminal amino groups and result in the need for altered coupling strategies for completion of analog synthesis.