Refolding of the mixed disulfide of bovine trypsinogen and glutathione.

The mixed disulfide of bovine trypsinogen and glutathione refolded with high yields at protein concentrations of 20 microgram/ml or less, at 4-25 degrees C, pH 8.0 to 8.7, in the presence of 3 to 6 mM cysteine under anaerobic conditions. The regenerated protein behaved as native trypsinogen as judged by gel exclusion chromatography, isoelectric focusing, and activation with bovine enterokinase or trypsin. However, refolded samples that were quenched with iodoacetate and analyzed by disc gel electrophoresis formed two components corresponding to trypsinogen and S-(carboxymethylcysteine)2-(179-203)-trypsinogen. The use of cysteine as a disulfide interchange catalyst caused reduction of the 179 to 203 disulfide bond, and quenching of the refolding mixture with iodoacetate produced the carboxymethylated derivative. The overall yield of the regenerated product was 70% and the half-time at 4 degrees C was 55 min.     

The renaturation of reduced chymotrypsinogen A in guanidine HCl. Refolding versus aggregation

The refolding and reoxidation of fully reduced and denatured chymotrypsinogen A have been studied in the presence of low concentrations of guanidine HCl or urea. Renaturation yields of 60 to 70% were observed when the reoxidation was facilitated by mixtures of reduced and oxidized glutathione. Refolding occurred within a narrow range of denaturant concentration (1.0 to 1.3 M guanidine HCl and 2 M urea) in which the native protein was shown to be stable, and the reduced protein was shown to regain the correct disulfide pairing. Renatured chymotrypsinogen is indistinguishable from the native zymogen in chromatographic behavior, potential chymotryptic activity, sedimentation coefficient, and spectral properties. The kinetics of renaturation were determined. Some of the protein species obtained at various times of renaturation were characterized as incorrectly oxidized molecules which could be renatured by thiol-catalyzed interchange of disulfide bonds.     

Refolding of sepharose-bound trypsinogen with disulfide 179 to 203 reduced and carboxymethylated

Disulfide 179 to 203 of native bovine trypsin was reduced with sodium borohydride and converted to the S-carboxymethyl derivative. The modified zymogen was attached to CNBr-activated Sepharose, and the resulting immobilized protein was used in refolding studies. The fully reduced protein was kept at 35°, at pH 8.5, under aerobic conditions, in a mixture of reduced and oxidized glutathione, until the sulfhydryl groups were reoxidized. A maximum yield of 55% was found for the regeneration of S-(carboxymethyl)₂-trypsinogen, and the activated product, S-(carboxymethyl)₂-trypsin, reacted with an active site reagent and gave the expected specific activity toward a typical trypsin substrate. Apparently, the refolding of immobilized S-(carboxymethyl)₂-trypsinogen regenerated the native structure of trypsinogen even though one of the six disulfides could no longer be formed.     

Detection of intermediate species in the refolding of bovine trypsinogen

The mixed disulfide of bovine trypsinogen and glutathione was refolded at pH 8.6 and 4 degrees C with a mixture of 3 mM cysteine and 1 mM cystine catalyzing disulfide interchange. The folding process was monitored by analysis of quenched samples with isoelectric focusing and size-exclusion chromatography. Isoelectric focusing showed a progressive change from a pI of 5.2 for the mixed disulfide derivative to a pI of 9.3 for native trypsinogen. A number of principal intermediates were detected as a function of the refolding time. These intermediates were also separated and further characterized by size-exclusion chromatography on columns of TSK G2000 SW operated in the high-performance liquid chromatographic mode. Rechromatography of a series of sequential fractions taken from the parental peak was necessary to resolve and characterize the principal intermediates. The loss of glutathione moieties produced a partly folded structure with an apparent hydrodynamic volume (Stokes radius, Rs) of 33.9 A. These structures became compact with time, and more intermediates were detected between 33.9 and 29.2 A. Finally, a change in conformation, resembling a two-state transition, changed the molecules of Rs 29.2 to the compact structure of native trypsinogen (22.4 A). The rate of formation of the native structure was determined from the progress curves derived from isoelectric focusing and size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Refolding of serine proteinases

Bovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%-50% at pH 8.6 and 4 degrees C, and the half-time for the refolding was approximately 60-75 min. We then refolded threonine-neochymotrypsinogen, which is a two-chain structure held together by disulfide bonds and produced on cleavage of Tyr 146-Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866-9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino- and carboxyl-terminal domains, were separated on Sephadex G-75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1:5 to 5:1 were refolded with yields of 21-28%. The lack of dependence on the concentration of either fragment and the relatively high yields suggest independent folding of the amino- and carboxyl-terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper geometry of the active site.     

Reoxidation of reduced bovine growth hormone from a stable secondary structure

In order to determine solution conditions appropriate for reoxidizing reduced bovine growth hormone (bGH), we have examined the possibility of using a particular denaturant concentration to poise the secondary and tertiary structure of the reduced protein in a stable, nativelike state. It was envisioned that the structure of the reduced molecule would differ from that of the final oxidized molecule solely by the absence of disulfide bonds. Dilution of concentrated samples of reduced and unfolded protein from 6.0 M guanidine into 4.5 M urea followed by air oxidation indicated it was possible to induce refolding and reoxidation to an oxidized monomeric species in high yield (approximately 90%). The choice of solution conditions was based on comparison of urea equilibrium denaturation data for native oxidized protein to those for completely reduced protein and to protein in which sulfhydryl groups had been either partially or completely reduced and subjected to modification with iodoacetamide or methyl methanethiolsulfonate. The denaturation behavior of these species supports the existence of equilibrium folding intermediates for bovine growth hormone and demonstrates that chemical modification of the protein is capable of inducing differences in the denaturation behavior of these intermediates. The changes in the protein absorption spectrum and helix-related circular dichroism signal, along with direct titration of protein sulfhydryl groups, indicated that the refolding/reoxidation of bGH is a multistate process. The ordered nature of the kinetic changes in these probes during reoxidation indicates that disulfide formation is a sequential process, with little mispairing in 4.5 M urea, and that it proceeds through one or more obligatory kinetic folding events. The equilibrium denaturation behavior of the oxidized molecule and the various chemically modified forms, together with the reoxidation data, indicated that the protein maintains a high degree of secondary structure without intrachain disulfide bonds. The formation of these disulfide bonds is a discrete process which occurs after a framework of protein secondary structure is established.     

Chemical and physical properties of the disulfides of bovine neurophysin-II.

Bovine neurophysin-II is shown to be very susceptible to partial reduction in the absence of urea. Reduction of an average of one disulfide leads to major changes in conformation and disulfide optical activity, manifest in part by pronounced far-uv ellipticity changes, complete loss of the 248-nm ellipticity band, and a shift of the 278-nm ellipticity band to shorter wavelengths with loss of half its intensity; the reduction process generates a mixture of products and appears to be accompanied by disulfide interchange. The circular dichroism data indicate that the disulfide(s) most susceptible to reduction or interchange are either the principal contributors to the 248- and 278-nm ellipticity bands or that the optical activity of other disulfides is dependent on their integrity. Peptides that bind to the hormone-binding site of neurophysin-II protect against reduction. On reoxidation of partially reduced neurophysin-II there is only a partial return of the native circular dichroism spectrum and electrophoretic behavior. The percentage of native protein in samples reoxidized following different degrees of reduction was estimated by comparison of the circular dichroism spectra of these samples with those of the fractionated native and denatured components of monoreduced-reoxidized neurophysin. Under our reoxidation conditions, less than 50% native protein was found in monoreduced-reoxidized neurophysin and less than 10% native protein was found in completely reduced-reoxidized neurophysin. The results are interpreted with qualified reference to a model in which one or more disulfides are "strained" in the native state and in which the native protein is unstable relative to species in which the disulfides are differently paired.     

Reversible denaturation of disulfide-reduced ovalbumin and its reoxidation generating the native cystine cross-link.

The authors in a previous report (Klausner, R. D., Kempf, C., Weinstein, J. N., Blumenthal, R., and van Renswoude, J. (1983) Biochem. J. 212, 801-810) have argued that native folding of ovalbumin occurs during translation, but not in a renaturation system of the denatured form. To re-examine the possibility, we searched for the conditions of correct oxidative refolding of denatured disulfide-reduced ovalbumin. Data of trypsin resistance, CD-spectrum, and selective reactivity of cysteine sulfhydryls revealed that the fully denatured protein can refold into the native conformation under disulfide-reduced conditions. The interconversion between the native and denatured forms was fully reversible with a free energy change for unfolding of 6.6 kcal/mol at 25 degrees C. Subsequent reoxidation under a variety of redox conditions generated only one disulfide bond in the reduced refolded protein with six cysteine sulfhydryls. Furthermore, the regenerated disulfide was found by peptide analyses to correspond to the native disulfide pairing, Cys73-Cys120. We, therefore, concluded that co-translational folding, if any, is not requisite for the correct oxidative folding of ovalbumin.     

Reduction and reoxidation of the neurotoxin II from the scorpion Androctonus australis Hector

Reoxidation of the totally reduced scorpion neurotoxin II from Androctonus australis Hector (four disulfide bridges) has been investigated. The totally reduced toxin was highly insoluble in neutral and alkaline conditions, which prevented the use of the usual air oxidation process for renaturation. We tested a new method in which the reduced molecules were first solubilized in 10% (v/v) acetic acid and then oxidized by air through dialysis against a series of buffers with a slow pH gradient from 2.2 to 7.0 or 8.0. In this system, up to 95% of the protein was recovered in solution. Addition of reduced and oxidized glutathione accelerated refolding and also permitted a better recovery of fully active peptide as measured by both toxicity to mice and ability to displace 125I radiolabeled toxin II from its binding site on rat brain synaptosomal fractions. The reoxidation reaction could also be monitored directly by high pressure liquid chromatography. A strong effect of guanidine hydrochloride concentration as well as the temperature was observed both on the solubility of the reoxidation intermediates and on the refolding pathway. Finally, the method used, i.e. dialysis reoxidation with a pH gradient from 2.2 to 8.0 in 0.1 M sodium phosphate, 0.1 M sodium chloride, 20 mM guanidine hydrochloride, 1 mM oxidized and reduced glutathione allowed regeneration in high yield (70%) of a reoxidized toxin form indistinguishable from the native toxin. A minor stable and inactive molecular species (about 30%) showing a difference in mobility by electrophoresis was also detected.     

Size-exclusion high performance liquid chromatography of native trypsinogen, the denatured protein, and partially refolded molecules. Further evidence that non-native disulfide bonds are dominant in refolding the completely reduced protein

Size-exclusion high performance liquid chromatography was used to compare the Stokes radius of the mixed disulfide of trypsinogen refolded for 10 min with the Stokes radius of denatured trypsinogen in high concentrations of urea. After folding for 10 min, rechromatography of a collection of sequential fractions of an initial separation showed that the fractions display microheterogeneity as seen in the value of the Stokes radius of each fraction. These intermediate species differed in their Stokes radius, and each had a globular structure cross-linked by disulfide bonds. In contrast, when trypsinogen with the native disulfides intact was equilibrated at different concentrations of urea (0-8 M), a progressive increase in Stokes radius was observed with extent of unfolding. Rechromatography of a series of fractions collected at a specific urea concentration showed that each had the same Stokes radius as the fraction in the initial separation. Urea-denatured trypsinogen and partially refolded trypsinogen must therefore differ in the disulfide pairing that links regions of the polypeptide chain. These observations support the suggestion that non-native disulfide bonds are responsible for the many stable conformations that form early in the folding of the mixed disulfide of trypsinogen (Light, A., and Higaki, J.N. (1987) Biochemistry 26, 5556-5564). These intermediates initially are loose structures (large Stokes radius) that become more compact with time (decreasing Stokes radius). The intermediates must therefore undergo a continuing disulfide interchange until native disulfides form late in the process when the stable conformation of the native molecule is reached.     

Disulfide-disulfide interchange catalyzed by a liver supernatant enzyme.

An enzyme widely distributed in rabbit tissues which catalyzes an interchange between N,N-di-dinitrophenyl-L-cystine and oxidized glutathione to form the mixed disulfide is described. D-Penicillamine disulfide can be substituted for oxidized glutathione and the mixed disulfide of cysteine and glutathione can serve as the sole substrate giving as one product of interchange, oxidized glutathione. The enzyme is very labile and only limited purification of it has been achieved. The activity increases with increasing pH above 6.6, the Km for N,N-di-dinitrophenyl-L-cystine is 0.2 mM and for oxidized glutathione 0.8 mM. The enzyme is inhibited by SH reagents with protection against iodoacetamide inactivation provided by N,N-di-dinitrophenyl-L-cystine. Evidence is presented that disulfide-disulfide interchange enzyme is a different activity from the previously described protein disulfide isomerase and thiol transferase.     

Proteolytic processing of presecretory proteins is required for development of biological activities in pancreatic exocrine proteins

The biological activities of pancreatic presecretory and secretory proteins synthesized in vitro were compared in studies of (a) the binding of nascent amylase to its substrate, glycogen, (b) the binding of nascent trypsinogen 1, trypsinogen 2+3, and chymotrypsinogen 1 to Sepharose-bound soybean trypsin inhibitor, and (c) the activation of nascent trypsinogen by porcine enterokinase. Nascent secretory proteins synthesized in vitro using a mRNA-dependent gel-filtered reticulocyte lysate translation system supplemented with canine pancreas rough microsomes or canine pancreas mRNA and micrococcal nuclease-treated microsomal membranes showed biological activities similar to authentic secretory proteins if oxidized glutathione was added during their synthesis. Proteins synthesized in the presence of membranes and the absence of glutathione showed significantly less biological activity due to incorrect development of conformation. Presecretory proteins synthesized in vitro with canine pancreas mRNA in the absence of microsomal membranes had little or no activity after translation in either the absence or presence of glutathione. These and previous findings (Scheele, G. A., and Jacoby, R. (1982) J. Biol. Chem. 257, 12277-12282) indicate that proteolytic removal of the NH2-terminal transport peptide is necessary to allow correct conformational development, including the formation of native disulfide bonds, which not only stabilizes the molecule but allows expression of authentic biological and probiological activity.     

Acceleration of disulfide-coupled protein folding using glutathione derivatives

Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives - glutamyl-cysteinyl-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (K(RCG) = 3.69 × 10(-3) s(-1)) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.     

Formation of the intrachain disulfide bond in the constant fragment of the immunoglobulin light chain

Regeneration by glutathione of the constant fragment of the immunoglobulin light chain was studied in the absence and presence of 8 m-urea. The species that appeared during the reaction of the reduced constant fragment with oxidized glutathione were trapped by alkylation with iodoacetamide and identified by electrophoresis in 15% polyacrylamide gel at pH 9.5. The kinetics of the reactions starting from various species formed during the reaction of the reduced constant fragment were also studied, and the overall reaction kinetics of the formation of the intrachain disulfide bond in the constant fragment were established in the absence and presence of urea.The reaction of the reduced constant fragment with oxidized glutathione was much slower but the yield of the constant fragment with the disulfide bond was much higher in the absence than in the presence of 8 m-urea. The slowness of the reaction in the absence of urea is due to the two cysteinyl residues of the reduced constant fragment being buried in the interior of the molecule and because oxidized glutathione is capable of reacting with the thiols only in the opened form of the protein molecule. The high yield is due to the cysteinyl thiol and the mixed disulfide in the intermediate forming an intrachain disulfide bond through thiol-disulfide interchange, the reaction sites being exposed to solvent and located at the appropriate proximity. These findings indicate first, that the appropriate proximity of a pair of cysteinyl residues is essential to form a disulfide bond and second, that they are not easily oxidized to disulfide if they are buried in the interior of the protein molecule.     

Solid-phase synthesis of bovine pancreatic trypsin inhibitor (BPTI) and two analogues. A chemical approach for evaluating the role of disulfide bridges in protein folding and stability.

The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Auxin-Modulated Protein Disulfide-Thiol-Interchange Activity from Soybean Plasma Membranes

The renaturation of scrambled (oxidized and inactive) RNase A is catalyzed by soybean (Glycine max cv Williams 82) plasma membranes. The catalysis is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid or by the natural auxin indole-3-acetic acid. The inactive auxin analog, 2,3-dichlorophenoxyacetic acid, is without effect. The activity occurs in the absence of external electron acceptors or donors and therefore appears to be a true disulfide-thiol-interchange activity between protein disulfides and thiols of RNase A and those of plasma membrane proteins. The activity is not affected by a mixture of reduced and oxidized glutathione. However, no auxin-stimulated activity was observed in the presence of either oxidized glutathione or reduced glutathione alone, a response characteristic of the previously described auxin-stimulated NADH oxidase activity of soybean plasma membranes. Taken together, the results suggest the operation in the plant plasma membrane of a protein disulfide-thiol-interchange activity that is stimulated by auxins. The auxin stimulations of the interchange activity are prevented by glutathione, reduced glutathione, and brefeldin A at concentrations that also prevent auxin stimulation of NADH oxidation by isolated plasma membranes and inhibit, as well, the auxin-stimulated elongation of excised segments of soybean hypocotyls.     

A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease.

Thioredoxin (Trx) from Escherichia coli was compared with bovine protein disulfide-isomerase (PDI) for its ability to catalyze native disulfide formation in either reduced or randomly oxidized (scrambled) ribonuclease A (RNase). On a molar basis, a 100-fold higher concentration of Trx than of PDI was required to give the same rate of native disulfide formation measured as recovery of RNase activity. A Pro-34 to His (P34H Trx) mutation in the active site of E. coli Trx (WCGPC), mimicking the two suggested active sites in PDI (WCGHC), increased the catalytic activity in disulfide formation about 10-fold. The mutant P34H Trx displayed a 35-mV higher redox potential (E'0) of the active site disulfide/dithiol relative to wild type Trx, making it more similar to the redox potential observed for PDI. This higher redox potential correlates well with the enhanced activity and suggests a role for the histidine side chain. Enzymatic isomerization of disulfides in scrambled, oxidized RNase requires the presence of a catalytic thiol such as GSH to initiate the thiol-disulfide interchange. Bovine thioredoxin reductase, together with NADPH, could replace GSH. For oxidative folding of reduced RNase in air with Trx, P34H Trx, or PDI, catalytic amounts of sodium selenite (1 microM) resulted in rapid disulfide formation and high yields of ribonuclease activity equivalent to previously known redox buffers of GSH and GSSG. These results demonstrate no obligatory role for glutathione in disulfide formation. A possible mechanism for the unknown thiol oxidative process accompanying folding and protein disulfide formation in vivo is discussed.     

Refolding of bovine threonine-neochymotrypsinogen

The mixed disulfide derivative of fully reduced neochymotrypsinogen was refolded at pH 9.2 and 4 degrees C with 4 mM cysteine as the disulfide interchange catalyst. The yield of regenerated neochymotrypsinogen was 25%; the corresponding yield of refolded chymotrypsinogen was 50%. The refolded neochymotrypsinogen exhibited the characteristics of the native molecule as determined from polyacrylamide gel electrophoresis and the enzymatic properties of the activated zymogen. The rate of refolding of neochymotrypsinogen was approximately the same as that found for chymotrypsinogen. These studies show that two separate fully reduced polypeptide chains were capable of refolding, associating with one another, and regenerating a native structure with full biological activity.     

Mixed disulfide with glutathione as an intermediate in the reaction catalyzed by glutathione reductase from yeast and as a major form of the enzyme in the cell

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH. The FAD of the reductase is reduced by NADPH, and reducing equivalents are passed to a redox-active disulfide to complete the first half-reaction. The nascent dithiol of two-electron reduced enzyme (EH(2)) interchanges with glutathione disulfide forming two molecules of glutathione in the second half-reaction. It has long been assumed that a mixed disulfide (MDS) between one of the nascent thiols and glutathione is an intermediate in this reaction. In addition to the nascent dithiol composed of Cys(45) and Cys(50), the enzyme contains an acid catalyst, His(456), having a pK(a) of 9.2 that protonates the first glutathione (residue numbers refer to the yeast enzyme sequence). Reduction of yeast glutathione reductase by glutathione and reoxidation of EH(2) by glutathione disulfide indicate that the mixed disulfide accumulates, in particular, at low pH. The reaction of glutathione disulfide with EH(2) is stoichiometric in the absence of an excess of glutathione. The equilibrium position among E(ox), MDS, and EH(2) is determined by the glutathione concentration and is not markedly influenced by pH between 6.2 and 8.5. The mixed disulfide is the principal product in the reaction of glutathione with oxidized enzyme (E(ox)) at pH 6. 2. Its spectrum can be distinguished from that of EH(2) by a slightly lower thiolate (Cys(50))-FAD charge-transfer absorbance at 540 nm. The high GSH/GSSG ratio in the cytoplasm dictates that the mixed disulfide will be the major enzyme species.