Ameloblastin fusion protein enhances pulpal healing and dentin formation in porcine teeth.

Ameloblastin (Ambn, also named "amelin" or "sheathlin") is a protein participating in enamel formation and mesenchymal-ectodermal interaction during early dentin formation in developing teeth. Experiments have demonstrated an association between Ambn expression and healing of acute pulp wounds. The purpose of this study was to investigate if local application of recombinant fusion Ambn (rAmbn) could influence reparative dentin formation in pulpotomized teeth. In this randomized, double-blinded study, pulpotomy was performed in 28 lower central incisors in 17 adult miniature pigs. Following the surgical procedure, the exposed pulp tissue was covered either with rAmbn or with calcium hydroxide. After 2, 4, or 8 weeks, the teeth were extracted and examined by histomorphometry and immunohistochemistry using antibodies against porcine ameloblastin, collagen type I, and dentin sialoprotein (DSP). In rAmbn-treated teeth, a substantial amount of newly formed reparative dentin was observed at the application site, completely bridging the pulpal wound. Dentin formation was also observed in calcium hydroxide-treated teeth; however, the amount of reparative dentin was significantly smaller (P < 0.001) than after rAmbn treatment. Immunohistochemistry confirmed that the new hard tissue formed was similar to dentin. This is the first time a direct link between ameloblastin and dentin formation has been made in vivo. The results suggest potential for rAmbn as a biologically active pulp-dressing agent for enhanced pulpal wound healing and reparative dentin formation after pulpotomy procedures.

Immunohistochemical characterization of rapid dentin formation induced by enamel matrix derivative.

The purpose of this study was to examine the pulpal expression of dentin-related proteins during enamel matrix derivative (EMD)-induced reparative dentin formation in a pulpotomy model in pig incisors. Pulpotomies were performed on 72 lower incisors in 24 adult miniature swine. The exposed pulp tissue was treated with EMD or covered with a calcium hydroxide paste (Dycal). At predefined time-points, ranging from 4 days to 12 weeks, experimental teeth were extracted and examined by use of light microscopy, and expression of dentin-related proteins in the pulps was investigated by immunohistochemistry, using antibodies against type I collagen, dentin sialoprotein (DSP), sheathlin, and EMD. In all EMD-treated teeth a substantial amount of reparative dentin formation was observed. The amount of reparative dentin in calcium hydroxide-treated teeth was significantly smaller than in EMD-treated teeth (P < 0.005) and was less effective in bridging the pulpal wounds. Immunohistochemistry demonstrated that enamel matrix proteins were present in detectable amounts at the application site for about 4 weeks. Moreover, the expression of proteins related to dentin formation in the wounded pulp tissue was about 2 weeks advanced in EMD-treated teeth. These findings demonstrate that enamel matrix molecules have the capacity to induce rapid pulpal wound healing in pulpotomized teeth, and suggest that the longevity and continued presence of enamel matrix macromolecules at the application site can be utilized to stimulate growth and repair of dentin over a period consistent with a favorable clinical outcome.

Expression of amelin and trauma-induced dentin formation.

According to recent studies, amelin (ameloblastin, sheathlin) is expressed in young odontoblasts at the initiation of dentin formation during odontogenesis. The purpose of the present investigation was to study whether amelin is also expressed at the onset of trauma-induced reparative dentin formation. The mandibular developing first molars of 5-day-old rats were surgically taken out, and their pulp tissue briefly separated from the inner dentin surface and immediately repositioned. Then the teeth were re-implanted in their alveoli. At 0, 2, 4, 6, 8, 12 or 14 days after surgery, the animals were sacrificed and the experimental teeth evaluated by histology and immunohistochemistry for amelin. At 2, 4, 6 and 8 days after surgery, the detached and traumatized odontoblasts in the experimental teeth exhibited increasing signs of degeneration and loss of intracellular structures. At days 6 and 8 after surgery, immunohistochemistry revealed a strong staining for amelin in the traumatized odontoblastic layer. Twelve and 14 days after replantation, only necrotic cell remnants of the traumatized odontoblasts were discernible. At this stage, no amelin could be detected by immunostaining. A wide zone of an unorganized mineralized tissue surrounded the odontoblastic cell remnants. On the pulpal side of the unorganized tissue, a new, highly organized tubular reparative dentin layer was observed, bordered by columnar odontoblast-like cells abutting on newly formed predentin. The results indicate that the initiation of trauma-induced reparative dentin formation mimics that of primary dentin formation and that amelin seems to be involved in both processes, possibly as a signaling molecule.

Amelin extracellular processing and aggregation during rat incisor amelogenesis.

Amelin (also known as ameloblastin and sheathlin) is a recently described protein that is secreted by ameloblasts during enamel formation. Here, the extracellular distribution and processing of amelin during rat incisor amelogenesis were investigated by Western blot probing using anti-recombinant rat amelin antibodies. In addition, the solubility behaviour and aggregative properties of rat amelin were investigated using a sequential extraction procedure involving (1) extraction with simulated enamel fluid to extract proteins most likely to be soluble in vivo; (2) extraction with phosphate buffer to desorb proteins bound to enamel crystal surfaces; (3) extraction with sodium dodecyl sulphate (SDS) to extract proteins present as insoluble aggregates; followed by (4) a final acid demineralization step to release any remaining proteins. Proteins immunoreactive to the anti-amelin antibodies were detectable in secretory- and transition-stage enamel. Maturation-stage enamel appeared devoid of amelin. The largest immunoreactive protein detected migrated at 68 kDa on SDS gels, corresponding to the M(r) of nascent amelin. Other immunoreactive bands at 52, 40, 37, 19, 17, 16, 15, 14 and 13 kDa were presumably amelin processing products. The sequential extraction procedure revealed that the 68-, 52-, 40-, 37- and 13-kDa amelins were completely extracted under solution conditions similar to those reported to exist in vivo. In contrast, the 19-, 17- and 16-kDa amelins were only partially extracted, whilst the 15- and 14-kDa amelins could not be extracted with simulated enamel fluid. A proportion of the remaining 17- and 16-kDa amelins was desorbed from the enamel crystals with phosphate buffer and appeared to have been mineral-bound. The 15- and 14-kDa amelins and the remainder of the 17- and 16-kDa amelins were extracted with SDS only, suggesting that these species were present in vivo as an insoluble aggregate. The results provide additional information on amelin processing and degradation, and on how such processing influences the solubility and aggregative properties of amelin-derived proteins.

Sequential expression of an amelin gene in mesenchymal and epithelial cells during odontogenesis in rats.

Novel mRNA isoforms encoding the enamel matrix proteins amelin-1, amelin-2 and ameloblastin have been recently described. We have applied detailed immunohistochemical as well as non-radioactive in situ hybridization analyses to follow amelin-1 expression in developing rat incisors and molars. We constructed an expression vector, overproduced recombinant amelin in Escherichia coli and prepared an antibody. In addition to the previously reported amelin mRNA expression patterns in ameloblasts, the amelin message was also detected in pulpal mesenchymal cells including preodontoblasts and young odontoblasts. The signal in these cells persisted until deposition of mantle dentin became evident. The immunolocalization of amelin-1 in preodontoblasts and ameloblasts essentially followed the pattern of mRNA expression. The most intense staining was found in the enamel matrix adjacent to secretory ameloblasts. Focal accumulations of immunoreactive material were found at the dentinoenamel junction during the maturation stage. Also, using 5'-RACE (Rapid Amplification of cDNA Ends) we could confirm only amelin-1 and ameloblastin messages in the total RNA pool from rat molars and conclude that amelin-2 is a truncated form of ameloblastin. The sequential expression of amelin in mesenchymal and epithelial cells suggests it plays a role in cell differentiation during early tooth development.

Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57.

To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S2) and reduced [Trx-(SH)2] forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt. In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations. In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the kcat/Km value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold. The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin. At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5--10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve. NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region. The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pKa of 7.5 in wt Trx-S2 as Asp 26. A profound change in the pKa of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2. The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins. A single pKa shifted to higher values is observed on both the Cys 32 and Cys 35 Cbeta resonances. Ultraviolet absorbance measurements (A240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9. The mutant proteins each show a single transition in the A240 measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results. The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two. It is clear that both thiol pK(a)s have been significantly shifted by the mutations. The Cys 32 pKa is moved from 7.1 in wt to 7.8-8.0 in the mutants. The value of the Cys 35 pKa either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance measurements or else is shifted to much higher pHs (> 10) where its transition is masked in both UV and NMR measurements by the effects of ionization of the tyrosine residues and unfolding of the protein. Our results strongly suggest that the buried Asp 26 carboxyl and Lys 57 epsilon-amino groups significantly affect the pK(a)s of the active site thiols, particularly that of the exposed low-pKa thiol Cys 32, thereby enhancing the rates of thiol-disulfide reactions at physiological pH.

Expression patterns of RNAs for amelin and amelogenin in developing rat molars and incisors.

We have recently identified a novel RNA sequence in ameloblasts, coding for amelin (Cerny et al., 1996). In the present paper, its expression has been compared with that of amelogenin in developing incisors and molars of rats, by means of in situ hybridization of paraffin sections. The RNAs for both amelin and amelogenin were highly expressed in secretory ameloblasts. The expression of RNA for amelogenin gradually decreased in the post-secretory ameloblasts. In contrast, the RNA expression for amelin remained high in post-secretory ameloblasts up to the stage of fusion between dental and oral epithelia at the time of tooth eruption. We suggest that amelin might be involved in the mineralization of enamel or in the attachment of ameloblasts to the enamel surface. The whole-mount in situ hybridization procedure is described for the first time in dental research. It proved to be a useful method and confirmed the results of the conventional in situ hybridization.

A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains.

Two variants of an mRNA sequence are identified that are expressed at high levels in rat ameloblasts during the formation of the enamel matrix. The sequences contain open reading frames for 407 and 324 amino acid residues, respectively. The encoded proteins, which we call amelins, are rich in proline, glycine, leucine, and alanine residues and contain the peptide domain DGEA, an integrin recognition sequence. The sequences coding for the C-terminal 305 amino acid residues, the 3' nontranslated part, and a microsatellite repeat at the nontranslated 5' region are identical in both mRNA variants. The remaining 5' regions contain 338 nucleotides unique to the long variant, 54 common nucleotides, and 46 nucleotides present only in the short variant. Eleven nucleotides have the potential to code for 5 amino acids of both proteins in different reading frames. The reading frame of the longer variant includes codons for a typical N-terminal signal peptide. The amelins are likely to be constituents of the enamel matrix and the only proteins that have so far been implicated in binding interactions between the ameloblast surface and its extracellular matrix.

Amelin: an enamel-related protein, transcribed in the cells of epithelial root sheath.

Since 1974, when Slavkin and his collaborators proposed the epithelial origin of cementum, many experiments have been carried out to provide evidence for deposition of enamel-related proteins along the root surface. However, neither amelogenin nor other proteins have fully satisfied expectations. In previous studies, we have identified a novel mRNA coding for an extracellular-like protein which we called amelin. It was expressed at high levels in secretory and postsecretory ameloblasts in rat molars and incisors. In situ hybridization experiments described in the present study also localized the amelin message to epithelial cells adjacent to the peripheral surface of newly deposited dentin in the root end and to cells embedded in cellular cementum in molars. In incisors, the amelin RNA positive cells were detected in the area where cementum formation had been initiated. No amelogenin RNA signal was found in the cells at the root surface. We postulate that the epithelial cells of the root sheath as well as the ameloblasts are synthesizing amelin which might be one of the key proteins coupled to the process of cementogenesis.

Replacement of Trp28 in Escherichia coli thioredoxin by site-directed mutagenesis affects thermodynamic stability but not function.

Escherichia coli thioredoxin contains two tryptophan residues (Trp28 and Trp31) situated close to the active site disulfide/dithiol. In order to probe the structural and functional roles of tryptophan in the mechanism of E. coli thioredoxin (Trx), we have replaced Trp28 with alanine using site-directed mutagenesis and expressed the mutant protein W28A in E. coli. Changes in the behavior of the mutant protein compared with the wild-type protein have been monitored by a number of physical and spectroscopic techniques and enzyme assays. As expected, removal of a tryptophan residue causes profound changes in the fluorescence spectrum of thioredoxin, particularly for the reduced protein (Trx-(SH)2), and to a lesser extent for the oxidized protein (Trx-S2). These results show that the major contribution to the strongly quenched fluorescence of Trx-S2 in both wild-type and mutant proteins is from Trp31, whereas the higher fluorescence quantum yield of Trx-(SH)2 in the wild-type protein is dominated by the emission from Trp28. The fluorescence, CD, and 1H NMR spectra are all indicative that the mutant protein is fully folded at pH 7 and room temperature, and, despite the significance of the change, from a tryptophan in close proximity to the active site to an alanine, the functions of the protein appear to be largely intact. W28A Trx-S2 is a good substrate for thioredoxin reductase, and W28A Trx-(SH)2 is as efficient as wild-type protein in reduction of insulin disulfides. DNA polymerase activity exhibited by the complex of phage T7 gene 5 protein and Trx-(SH)2 is affected only marginally by the W28A substitution, consistent with the buried position of Trp28 in the protein. However, the thermodynamic stability of the molecule appears to have been greatly reduced by the mutation: guanidine hydrochloride unfolds the protein at a significantly lower concentration for the mutant than for wild type, and the thermal stability is reduced by about 10 degrees C in each case. The stability of each form of the protein appears to be reduced by the same amount, an indication that the effect of the mutation is identical in both forms of the protein. Thus, despite its close proximity to the active site, the Trp28 residue of thioredoxin is not apparently essential to the electron transfer mechanism, but rather contributes to the stability of the protein fold in the active site region.

Expression of amelogenin mRNA sequences during development of rat molars.

The expression of amelogenin mRNA in growing rat molars was studied. Northern blotting and the analysis of cDNA isolates revealed two predoninant variants. One group of cDNA inserts contained sequences of a long mRNA version and the other group contained mRNA sequences of the shorter leucin-rich amelogenin polypeptide (LRAP). The LRAP group was deficient in an internal stretch which coded for a peptide with a high potential for beta turns. Northern blot experiments showed that most amelogenin RNA in rat teeth was represented by two bands of 1.1 and 0.8 kb. Two oligonucleotide probes were designed that were specific for the long version and for the LRAP variant. The probes were used for in situ hybridization experiments on sections of developing maxillar teeth of rats between day 2 and day 15 after birth. Both RNA species were accumulated concomitantly and exclusively in cells of the inner enamel epithelium. Expression was first observed at the mesial cusp sides and finally involved the whole ameloblast layer except for the cells adjacent to the enamel-free region at the tip of the cusps. The early amelogenin RNA expression occurred adjacent to the initial deposition of the dentin matrix. Low amounts of amelogenin RNA persisted after the differentiation of ameloblasts into the maturative stage. The sequence of events was similar in all three molars.

Rapid isolation of homogeneous cloned T7 gene 5 protein and T7 DNA polymerase by affinity chromatography on immobilized thioredoxin.

Phage T7 DNA polymerase consists of a strong 1:1 complex of T7 gene 5 protein (80 kDa) and the reduced form of Escherichia coli thioredoxin (12 kDa). Immobilization of E. coli thioredoxin on the agarose matrix Affi-Gel retained both its redox activity and its ability to bind T7 gene 5 protein. This was used to develop a simple and fast high-yield purification method. Cloned T7 gene 5 protein, expressed in a thioredoxin-negative host cell, was isolated in pure and highly active form after elution from Affi-Gel--thioredoxin with a pH gradient from 10 to 12. This purification step separated gene 5 protein from variable amounts of two sets of reconstituting large polypeptide fragments without catalytic activity. Proteolytic cleavage in vivo probably gave rise to the fragments, the generation of which was mimicked by trypsin cleavage of pure gene 5 protein. The gene 5 protein preparation had an inherent low DNA polymerase and double-stranded 3'-exonuclease activity, which was stimulated at least 30-fold by the presence of reduced thioredoxin. Highly active and pure T7 DNA polymerase was obtained by reconstitution of gene 5 protein with thioredoxin and was isolated by phosphocellulose or FPLC Mono Q chromatography. The gene 5 protein and T7 DNA polymerase preparations are suitable for further physicochemical characterization and as reagents in DNA sequencing.

Thioredoxin reductase-dependent insulin disulfide reduction by phage T7 DNA polymerase reflects dissociation of the enzyme into subunits.

Phage T7 DNA polymerase contains Escherichia coli thioredoxin as a subunit and is a 1:1 complex with T7 gene 5 protein. The enzyme showed high thioredoxin activity in assays at 37 degrees C using reduction of insulin disulfides with NADPH and thioredoxin reductase, leading Randahl (Randahl, H. (1982) FEBS Lett. 150, 109-113) to propose that the thioredoxin dithiol active site is exposed in T7 DNA polymerase. However, T7 DNA polymerase and free thioredoxin differ in reactivity with iodoacetic acid after preincubation with dithiothreitol or incubation with insulin. Insulin reduction assays work at low temperatures even at 0 degrees C. The time and temperature dependence of the thioredoxin activity of T7 DNA polymerase demonstrated that dissociation into subunits at 25 or 37 degrees C accounts for the previously observed activity. Thus, T7 DNA polymerase contains the reduced form of thioredoxin with its active site SH groups masked by the subunit contact with the gene 5 protein in agreement with the results of Adler and Modrich (Adler, S., and Modrich, P. (1983) J. Biol. Chem. 258, 6956-6962). The subunit interaction of thioredoxin and gene 5 protein is salt-insensitive, but markedly temperature-dependent consistent with involvement of a hydrophobic surface area in reduced thioredoxin.

T7 DNA polymerase is not a zinc-metalloenzyme and the polymerase and exonuclease activities are inhibited by zinc ions.

Phage T7 DNA polymerase purified to homogeneity by an antithioredoxin immunoadsorbent technique was resolved into its active subunits the gene 5 protein and Escherichia coli thioredoxin by a novel technique involving chromatography on Sephadex G-50 at pH 11.5. Analysis of the metal content of the holoenzyme by atomic absorption spectroscopy showed that it did not contain stoichiometric amounts of zinc. Determination of polymerase and exonuclease activities of the holoenzyme and the gene 5 protein in assay mixtures containing enzyme concentrations in excess of the Zn2+ concentration showed full activity. Addition of Zn2+ resulted in no stimulation and the activities were completely inhibited by 0.1 mM Zn2+. These results demonstrate that the essential T7 DNA polymerase is not a zinc-metalloenzyme and suggest that DNA polymerases show no functional requirement for Zn2+.

An improved purification method and a physical characterization of phage T7 DNA polymerase.

The immunoadsorbent used to purify T7 DNA polymerase contains antibodies directed towards thioredoxin. Elution of the enzyme is made by a pulse of buffer at pH 12.0. This decreases the binding capacity of the column. Binding experiments with [3H]thioredoxin showed that the effect was caused by reduction of the antibodies by thiols in alkaline buffers. T7 DNA polymerase aggregated and irreversibly lost activity in buffers of low ionic strength. Experiments with gel chromatography and glycerol density gradient centrifugation showed that 0.2 M sodium chloride was required to keep the enzyme in its monomeric form. The sedimentation coefficient and the Stokes' radius are 5.3 S and 4.6 nm respectively, evaluated by gel chromatography and glycerol density gradient centrifugation techniques. The frictional ratio of 1.49 indicates that the T7 DNA polymerase is an asymmetrical protein.

Characterization of bacteriophage T7 DNA polymerase purified to homogeneity by antithioredoxin immunoadsorbent chromatography.

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-coded thioredoxin. We have purified T7 DNA polymerase to homogeneity from T7-infected Escherichia coli B cells with a novel technique based on immunoadsorbent affinity chromatography. The enzyme binds quantitatively to a column of anti-thioredoxin Sepharose 4B and remains as an active complex in the immobilized state. It is eluted in fully active and highly purified form by a pulse of buffer at pH 12. After a final phosphocellulose chromatography, T7 DNA polymerase of better than 99% purity, as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis, is obtained. Determination of the molecular weight by sedimentation equilibrium centrifugation gives a value of 112,000. Denaturing gels showed that the enzyme is composed of gene 5 protein (Mr = 87,000 +/- 3,000) and thioredoxin (Mr = 12,000) in a 1:1 stoichiometry. The amino acid composition of the enzyme and its spectrum was determined. The DNA polymerase activity is dependent on sulfhydryl compounds, sensitive to salt, and shows a comparatively high Km value for the four deoxyribonucleotides. The enzyme preparation has an inherent 3' leads to 5' exonuclease activity, attacking both native and denatured T7 DNA; it is free from detectable endonuclease activity.

Structure and enzymatic functions of thioredoxin refolded by complementation of two tryptic peptide fragments.

The physicochemical and catalytic properties of thioredoxin-T' are described. This complemented protein structure consists of a 1:1 complex between the inactive fragments thioredoxin-T-(1--73) and thioredoxin T-(74--108). These are generated by selective trypsin cleavage at Arg-73 in lysine-modified and denatured Escherichia coli thioredoxin. Thioredoxin-T' was a slowly formed but stable complex with an apparent KD below 10(-8) M. The tryptophan fluorescence spectrum and the CD spectrum were very similar to those of native thioredoxin; some conformational differences were detected by gel chromatography and radioimmunoassay. Thioredoxin-T'-S2 was a substrate for NADPH and thioredoxin reductase and had 1--2% of the activity of native thioredoxin. This low relative activity was the result of a major increase in the Km value. Thioredoxin-(SH)2 was a hydrogen donor for E. coli ribonucleotide reductase with about 3% relative activity. These results for thioredoxin-T' are correlated with the known three-dimensional structure of thioredoxin. The microenvironment around Arg-73 that is close to the active disulfide appears to be of critical importance for the interactions of thioredoxin with thioredoxin reductase and ribonucleotide reductase.

Reconstitution of Escherichia coli thioredoxin from complementing peptide fragments obtained by cleavage at methionine-37 or arginine-73.

Thioredoxin from Escherichia coli (a small hydrogen transport protein containing 108 amino acid residues and having in its oxidized form a single disulfide bond) was acylated with citraconic anhydride. Citraconylation of all amino groups resulted in total loss of enzymatic activity with thioredoxin reductase and immunoprecipitin activity with antithioredoxin antibodies; both these activities were fully restored after deblocking of the citraconylated protein by acid treatment. Large enzymatically inactive peptide fragments of thioredoxin were prepared by selective cleavage at Arg-73 and Met-37, respectively, and tested for their antigenic activity with antibodies against native thioredoxin. Thioredoxin-T-(1-73) and thioredoxin-T-(74-108) were separated by Sephadex G-50 chromatography in 50% acetic acid of a deblocked trypsin digest of citraconylated thioredoxin. Thioredoxin-T-(1-73) afforded about 25% of the corresponding immunoprecipitate of native thioredoxin without significant inhibition at antigen excess. Thioredoxin-T-(74-108) gave no immunoprecipitate but was a potent inhibitor of the reaction of thioredoxin and antithioredoxin as measured by turbidity formation. A major antigenic determinant of thioredoxin was present in the COOH-terminal sequence of the molecule. An equimolar mixture of thioredoxin-T-(1-73) and thioredoxin-T-(74-108) showed full immunoprecipitation activity with antithioredoxin and significant enzymatic activity with thioredoxin reductase. Gel chromatography experiments at pH 8 with the peptide mixture showed a main symmetrical peak with elution volume and amino acid composition identical with native thioredoxin. The results strongly suggested reconstitution of these two fragments to a complex, thioredoxin-T', with a conformation similar to native thioredoxin. Reconstitution of a thioredoxin-like structure was also obtained from a mixture of the overlapping fragments thioredoxin-T-(1-73) and thioredoxin-C-(38-108), although these peptides represented more than the 108 amino acid residues of the protein. Previous results showed reconstitution of thioredoxin-C-(1-37) and thioredoxin-C-(38-108) to a complex called thioredoxin-C' (Holmgren, A. (1972) Fed. Eur. Biochem. Soc. Lett. 24, 351-354). Together with the present results, this shows that three different combinations of two larger peptide fragments obtained by cleavage at two permissible sites gives reconstitution of thioredoxin. In each case, at least one of the component peptides showed strong immunochemical activity with antibodies to native thioredoxin, Netion from a tetrameric to a dimeri