Disulfide Exchange Reaction Research Articles

Preparation of poly(lactic acid)/siloxane/calcium carbonate composite membranes with antibacterial activity

A poly(lactic acid) (PLA)/siloxane/calcium carbonate composite membrane containing mercapto groups (PSC-SH) with antibacterial ability and excellent bone-forming ability was prepared using 3-mercaptopropyltrimethoxysilane for application in guided bone regeneration. Mercapto groups were reported to adsorb silver ions, which are well known to show antibacterial activity. Ionic silicon species were reported to stimulate the proliferation of osteoblasts. A PSC-SH membrane with a thickness of about 10 μm shows high flexibility. The PLA in PSC-SH was converted from the crystalline phase to the amorphous phase due to dispersion of condensed siloxane clusters. The amount of mercapto group on PSC-SH surface was estimated to be about 55 nmol mm −2 by quantitative analysis using the thiol–disulfide exchange reaction. PSC-SH adsorbed silver ions on its surface after being soaked in 6 μM silver acetate aqueous solution for 1 min. The adsorbed silver ions were seen by X-ray photoelectron spectroscopy to form SAg and SO 3Ag bonds. A trace amount of ionic silicon species was released from the membrane after soaking in culture medium. PSC-SH with adsorbed silver ions showed good antibacterial activity and cellular compatibility in tests conducted with Staphylococcus aureus and mouse osteoblast-like cells, respectively. Antibacterial activity is expected to occur during the implantation operation by the silver ions but not to remain in the body for a long period, as the ions were present on the surface of the membrane but not inside the structure. The membrane should be useful as a biodegradable material with antibacterial activity and bone-forming ability.

A novel approach to enhancing cellular glutathione levels

GSH and GSH-associated metabolism provide the major line of defense for the protection of cells from oxidative and other forms of toxic stress. Of the three amino acids that comprise GSH, cysteine is limiting for GSH synthesis. As extracellularly cysteine is readily oxidized to form cystine, cystine transport mechanisms are essential to provide cells with cysteine. Cystine uptake is mediated by system x(c)(-), a Na(+)-independent cystine/glutamate antiporter. Inhibition of system x(c)(-) by millimolar concentrations of glutamate, a pathway termed oxidative glutamate toxicity, results in GSH depletion and nerve cell death. Recently, we described a series of compounds derived from the conjugation of epicatechin (EC) with cysteine and cysteine derivatives that protected nerve cells in culture from oxidative glutamate toxicity by maintaining GSH levels. In this study, we characterize an additional EC conjugate, cysteamine-EC, that is 5- to 10-fold more potent than the earlier conjugates. In addition, we show that these EC conjugates maintain GSH levels by enhancing the uptake of cystine into cells through induction of a disulfide exchange reaction, thereby uncoupling the uptake from system x(c)(-). Thus, these novel EC conjugates have the potential to enhance GSH synthesis under a wide variety of forms of toxic stress.

Engineered extracellular matrices with cleavable crosslinkers for cell expansion and easy cell recovery

An unmet need for expansion of primary cells and progenitor cells in three dimensions (3-D) is a synthetic mimic of the extracellular matrix (ECM) with user-controllable composition that would permit rapid recovery of viable cells under mild, non-enzymatic conditions. Three block copolymers based on disulfide-containing polyethylene glycol diacrylate crosslinkers were synthesized, and were used to crosslink thiol-modified hyaluronan and gelatin macromonomers in the presence of cells. The triblock PEGSSDA contained a single disulfide-containing block, the pentablock PEG(SS) 2DA contained two disulfide blocks, and the heptablock PEG(SS) 3DA contained three disulfide blocks. For each hydrogel composition, four cell types were encapsulated in 3-D, and growth and proliferation were evaluated. Murine NIH 3T3 fibroblasts, human HepG2 C3A hepatocytes, human bone marrow-derived mesenchymal stem cells (MSCs), and human umbilical vein endothelial cells (HUVECs) all showed excellent viability and growth during expansion in 3-D in the three disulfide block copolymer crosslinkers. After cell expansion, the hydrogels were dissociated using the thiol–disulfide exchange reaction in the presence of N-acetyl-cysteine or glutathione, which dissolved the hydrogel network. After dissolution, cells were recovered in high yield and with high viability by gentle centrifugation.

Recent Developments in Disulfide Bond Formation

This review summarizes the recent developments of di-sulfidebond formation with a variety of reagents. The scope and limitationsof the presented methods are discussed. The syntheses of unsymmetricaldisulfides are highlighted in order to present the most versatileachievements. 1 Introduction 2 Preparation of Disulfides 2.1 By Oxidation of Thiols 2.2 By Reductive Coupling of Sulfonyl Chlorides 2.3 By Reaction with Sulfur Monochloride 2.4 By Radical Cyclization of Substituted Aminothiourea Derivatives- 2.5 Using 1-Chlorobenzotriazole 2.6 From Elemental Sulfur 2.7 From Organophosphorus Sulfenyl Bromide 2.8 From N-Trifluoroacetyl Arenesulfenamides 2.9 Using Phase-Transfer Catalysis 2.10 From a Thiol-Disulfide Exchange Reaction 2.11 From Thiocyanates 2.12 From 2-(Trimethylsilyl)ethyl Sulfides 2.13 From Thiosulfonates and Thiosulfates 2.14 From Thioesters 2.15 By Disulfide Exchange Reactions 2.16 From Tetrathiomolybdate 2.17 By Miscellaneous Reactions 3 Conclusions

Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

Thioredoxin A Active-Site Mutants Form Mixed Disulfide Dimers That Resemble Enzyme–Substrate Reaction Intermediates

Thioredoxin functions in nearly all organisms as the major thiol–disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme–substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.

A Trade-Off between Catalytic Power and Substrate Inhibition in TCHQ Dehalogenase

Tetrachlorohydroquinone (TCHQ) dehalogenase is profoundly inhibited by its aromatic substrates, TCHQ and trichlorohydroquinone (TriCHQ). Surprisingly, mutations that change Ile12 to either Ser or Ala give an enzyme that shows no substrate inhibition. We have previously shown that TriCHQ is a noncompetitive inhibitor of the thiol-disulfide exchange reaction between glutathione and ESSG, a covalent adduct between Cys13 and glutathione formed during dehalogenation of the substrate. Substrate inhibition of the thiol-disulfide exchange reaction is less severe in the I12S and I12A mutant enzymes, primarily due to weaker binding of TriCHQ to ESSG. These mutations also result in a decrease in the rate of dehalogenation. Because the rate-limiting step in the I12S and I12A enzymes is dehalogenation, rather than the thiol-disulfide exchange reaction, the relatively modest inhibition of the thiol-disulfide exchange reaction does not affect the overall rate of turnover.

Dynamic rearrangement of disulfide bridges influences solubility of whey protein coatings

In this study, the mechanism governing the solubility of whey protein films and coatings was investigated. The building blocks for these films and coatings were aggregated whey proteins. Solubility was related to the continuity of the network of disulfide cross links between the building blocks and the dynamics of rearrangements of disulfide bonds occurring via the so-called thiol–disulfide exchange reaction. By controlling either the accessibility of thiol groups or the accessibility of disulfide bonds within the whey protein aggregates, solubility of whey protein coatings and films could be tuned. These new insights will help improving the properties of protein coating, promoting the applicability of whey proteins in films, encapsulates and coatings.

Mechanism of Thiolate−Disulfide Interchange Reactions in Biochemistry

Both density functional theory (DFT) (B3LYP) and CCSD ab initio calculations were employed in a theoretical investigation of the mechanism of thiolate-disulfide exchange reactions. The reaction pathway for degenerate thiolate-disulfide exchange reactions with dimethyl disulfide has been shown to proceed through a SN2-like transition structure that is very close in energy to the corresponding trisulfur anionic intermediate ([delta-S-S-Sdelta(-)]). When relatively small substituents are involved, the level of theory must be increased to CCSD to make this rather subtle mechanistic distinction. With the more sterically hindered exchange reaction involving t-butyl mercaptide and di-t-butyl disulfide, the potential energy surface exhibits a distinct preference for the S(N)2 displacement pathway with an activation barrier of 9.8 kcal/mol. When corrections for solvent polarity are included (COSMO), an S(N)2 mechanism is also implicated in both polar and nonpolar solvents. DFT studies on thiolate-disulfide exchange, when the substituent is a model peptide, strongly support the intermediacy of a trisulfur intermediate that lies 10.7 kcal/mol below isolated reactants. A well depth of this magnitude should provide a sufficient lifetime of the intermediate to accommodate the requisite conformational adjustments that accompanies formation of the new disulfide bond.

Functional Characterization of Mia40p, the Central Component of the Disulfide Relay System of the Mitochondrial Intermembrane Space

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.

Protein Disulfide Isomerases fromC. elegansare Equally Efficient at Thiol-Disulfide Exchange in Simple Peptide-Based Systems But Show Differences in Reactivity Towards Protein Substrates

Although the formation of disulfide bonds is an essential process in every living organism, only little is known about the mechanisms in multicellular eukaryotic systems. The reason for this uncertainty is that in addition to the well-known key enzyme protein disulfide isomerase (PDI), several PDI-like proteins are present in the ER of metazoans. In total, there are now 18 PDI-family members in the human endoplasmic reticulum, with different domain architectures and active site chemistries. To understand why multicellular organisms express multiple proteins with similarity to the archetypal mammalian PDI, the properties of three PDIs from the nematode C. elegans were investigated. Here the authors demonstrate that PDI-1, PDI-2, and PDI-3 show comparable kinetic properties in catalyzing thiol:disulfide exchange reactions in two simple peptide-based assays. However, the three enzymes exhibited clear differences in their reactivity towards protein substrates. The authors therefore propose that the three PDIs can catalyze similar thiol-disulfide exchange reactions in a substrate, but due to differences in substrate binding, they can direct a folding polypeptide chain onto different folding pathways and hence fulfil distinct and different functions in the organism.

In vitro insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.

Facile Synthetic Procedure for ω, Primary Amine Functionalization Directly in Water for Subsequent Fluorescent Labeling and Potential Bioconjugation of RAFT-Synthesized (Co)Polymers

We describe a facile method to amine functionalize and subsequently fluorescently label polymethacrylamides synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. RAFT-generated poly(N-(2-hydroxypropyl) methacrylamide-b-N-[3-(dimethylamino)propyl] methacrylamide) (poly(HPMA-b-DMAPMA)), a water soluble biocompatible polymer, is first converted to a polymeric thiol and functionalized with a primary amine through a disulfide exchange reaction with cystamine and subsequently reacted with the amine-functionalized fluorescent dye, 6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl ester (5-SFX). Poly(HPMA258-b-DMAPMA13) (Mn = 39 700 g/mol, Mw/Mn = 1.06), previously synthesized by RAFT polymerization, was used to demonstrate this facile labeling method. The problem with labeling the omega-terminal chain end of a RAFT-synthesized polymethacrylamide is that the reduced end yields a tertiary thiol with low reactivity. The key to labeling poly(HPMA-b-DMAPMA) is to first reduce the dithioester chain end with a strong reducing agent such as NaBH4, and then functionalize the tertiary polymeric thiol with a primary amine through a disulfide exchange reaction with dihydrochloride cystamine. We show that the disulfide exchange reaction is efficient and that the amine-functionalized poly(HPMA-b-DMAPMA) can be easily labeled with the fluorescent dye, 5-SFX. This concept is proven by using a ninhydrin assay to detect primary amines and UV-vis spectroscopy to measure the degree of conjugation.

A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR

Oxidation of protein thiolates is central to numerous redox-regulated processes. Bacillus subtilis OhrR is an organic peroxide sensor that represses expression of an inducible peroxiredoxin, OhrA. Here, we present evidence that oxidation of the sole cysteine residue in OhrR leads to a sulfenic acid-containing intermediate that retains DNA-binding activity: further reaction to generate either a mixed disulfide (S-thiolation) or a protein sulfenamide (sulfenyl-amide) derivative is essential for derepression. Protein S-thiolation protects OhrR from overoxidation and provides for a facile regeneration of active OhrR by thiol-disulfide exchange reactions. The sulfenamide can also be reduced by thiol-disulfide exchange reactions, although this process is much slower than for mixed disulfides. Recovery of oxidized OhrR from B. subtilis identifies three distinct S-thiolated species, including mixed disulfides with a novel 398-Da thiol, cysteine, and CoASH. Evidence for in vivo formation of the sulfenamide derivative is also presented.

Synthesis and in Vitro Characterization of an ABC Triblock Copolymer for siRNA Delivery

The ability to specifically down-regulate gene expression using the RNAi pathway in mammalian cells has tremendous potential in therapy and in basic science. However, delivery systems capable of efficient and biocompatible delivery of siRNA to target cells are not yet satisfactory. Here, we report the synthesis and in vitro characterization of ABC triblock copolymers that self-assemble with siRNA based on electrostatics and with each other by hydrophobic interactions. The ABC triblock copolymer is based on poly(ethylene glycol) (PEG), poly(propylene sulfide) (PPS), and a positively charged peptide (PEG-PPS-peptide). The diblock copolymer PEG(45)-PPS(5,10) was synthesized using anionic polymerization of propylene sulfide upon a PEG macroinitiator, and the peptide domain was coupled to the PPS terminus using a disulfide exchange reaction with an N-terminal cysteine residue on the peptide. The peptides were designed to interact electrostatically with siRNA, selecting the TAT peptide domain of HIV (RKKRRQRRR) and an oligolysine (Lys(9)). The resulting triblock copolymers were able to self-assemble with siRNA as demonstrated by dynamic light scattering and gel electrophoresis. Complex size was found to be dependent on the amount of polymer used (charge ratio) and the length of the hydrophobic PPS block, achieving sizes ranging from 171 nm to 601 nm. Cell internalization and gene expression down-regulation studies showed that the triblock copolymers are able to transport siRNA inside the cell and mediate gene expression down-regulation, with the amount of internalization and gene transfer affected by charge ratio, PPS length, and the presence of serum. The proposed triblock was able to mediate gene expression down-regulation of GAPDH, achieving up to 90.5% +/- 0.02% down-regulation.

Cholesterol−Phospholipid Complexation in Fluid Bilayers as Evidenced by Nearest-Neighbor Recognition Measurements

Nearest-neighbor recognition experiments have been carried out using varying ratios of exchangeable dimer analogs of 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol and 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol in cholesterol-rich unilamellar vesicles at 60 degrees C. Equilibrium dimer distributions that were obtained support a structural model of the liquid-ordered bilayer in which free cholesterol and the longer-chain phospholipid homodimer are in equilibrium with a complex of unique stoichiometry, where one cholesterol molecule combines with two of the long-chain phospholipid homodimers. In this model, the mixing of the short-chain phospholipids with the uncomplexed long-chain phospholipids is ideal, and the complexed dimers are shielded from the disulfide exchange reaction.

Structures of Ternary Complexes of BphK, a Bacterial Glutathione S-Transferase That Reductively Dechlorinates Polychlorinated Biphenyl Metabolites

Prokaryotic glutathione S-transferases are as diverse as their eukaryotic counterparts but are much less well characterized. BphK from Burkholderia xenovorans LB400 consumes two GSH molecules to reductively dehalogenate chlorinated 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), inhibitory polychlorinated biphenyl metabolites. Crystallographic structures of two ternary complexes of BphK were solved to a resolution of 2.1A. In the BphK-GSH-HOPDA complex, GSH and HOPDA molecules occupy the G- and H-subsites, respectively. The thiol nucleophile of the GSH molecule is positioned for SN2 attack at carbon 3 of the bound HOPDA. The respective sulfur atoms of conserved Cys-10 and the bound GSH are within 3.0A, consistent with product release and the formation of a mixed disulfide intermediate. In the BphK-(GSH)2 complex, a GSH molecule occupies each of the two subsites. The three sulfur atoms of the two GSH molecules and Cys-10 are aligned suitably for a disulfide exchange reaction that would regenerate the resting enzyme and yield disulfide-linked GSH molecules. A second conserved residue, His-106, is adjacent to the thiols of Cys-10 and the GSH bound to the G-subsite and thus may stabilize a transition state in the disulfide exchange reaction. Overall, the structures support and elaborate a proposed dehalogenation mechanism for BphK and provide insight into the plasticity of the H-subsite.

The Interaction of Thioredoxin with Txnip: EVIDENCE FOR FORMATION OF A MIXED DISULFIDE BY DISULFIDE EXCHANGE

The thioredoxin system plays an important role in maintaining a reducing environment in the cell. Recently, several thioredoxin binding partners have been identified and proposed to mediate aspects of redox signaling, but the significance of these interactions is unclear in part due to incomplete understanding of the mechanism for thioredoxin binding. Thioredoxin-interacting protein (Txnip) is critical for regulation of glucose metabolism, the only currently known function of which is to bind and inhibit thioredoxin. We explored the mechanism of the Txnip-thioredoxin interaction and present evidence that Txnip and thioredoxin form a stable disulfide-linked complex. We identified two Txnip cysteines that are important for thioredoxin binding and showed that this interaction is consistent with a disulfide exchange reaction between oxidized Txnip and reduced thioredoxin. These cysteines are not conserved in the broader family of arrestin domain-containing proteins, and we demonstrate that the thioredoxin-binding property of Txnip is unique. These data suggest that Txnip is a target of reduced thioredoxin and provide insight into the potential role of Txnip as a redox-sensitive signaling protein.

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3β cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3β cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3β cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.

Investigating the interaction between ribonucleoside triphosphosphate reductase and thioredoxin in Lactobacillus leichmannii

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides (dNTPs) in all organisms and thus play an essential role in DNA biosynthesis and repair. This function makes RNRs attractive targets for anticancer and antiparasitic chemotherapies since inhibition of these enzymes disrupts the dNTP balance within the cell, leading to cell death. Previous work has shown that thioredoxin (Trx)/thioredoxin reductase systems are responsible for the re-reduction of class I and class II RNRs through disulfide exchange reactions involving conserved cysteine residues. To study this re-reduction reaction, we have selected proteins from Lactobacillus leichmannii, an ideal model system due to the relative simplicity of its ribonucleoside triphosphate reductase (RTPR) and the presence of only one Trx in its genome. We have cloned Trx from Lactobacillus leichmannii and purified the protein to homogeneity. To investigate which of the cysteine residues at the C-terminus of RTPR are more reactive toward Trx, we have prepared RTPR mutant proteins C731S and C736S. Further, to investigate which of the cysteines in the conserved Trx ‘CGPC’ motif are more reactive toward RTPR, we have prepared C28S and C31S Trx mutant proteins. Preliminary results of the disulfide-mediated cross-linking of RTPR with Trx will be presented. (Supported by National Institutes of Health GM65337)