Fluorescent Thiol Reagent Research Articles

ChemInform Abstract: Synthesis and Properties of a Maleimide Fluorescent Thiol Reagent Derived from a Naphthopyranone.

N-(2-Carbomethoxy-9-methoxy-3-oxo-3H-naphtho[2,1-b]pyran-10-yl)maleimide 2 was designed and synthesized as a new maleimide fluorescent thiol reagent. The optical properties of 2 were investigated with and without the addition of glutathione, GSH. We have found 2 is twice as sensitive as DACM and is of comparable sensitivity to CPM for detection of thiols. The emission maximum for the GSH adduct of 2 is 513 nm which is at longer wavelength than the GSH adducts of both DACM and CPM.

Dissecting the mechanism of Ca2+‐triggered membrane fusion: Probing protein function using thiol reactivity

1. Ca(2+)-triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage-specific native preparation fully enabling the directly coupled functional-molecular analyses necessary to identify critical components of fast triggered membrane fusion. 2. Recent work on lipidic components has established a direct role for cholesterol in the fusion mechanism via local contribution of negative curvature to readily enable the formation of transient lipidic fusion intermediates. In addition, cholesterol- and sphingomyelin-enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca(2+) transient. 3. There is less known about the identity of proteins involved in the Ca(2+)-triggering steps of membrane fusion. Thiol reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol-reactive reagents have identified different effects on Ca(2+) sensitivity and the extent of fusion, suggesting that there are at least two distinct thiol sites that participate in the fusion mechanism: one that regulates the efficiency of Ca(2+) sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca(2+) sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was used to potentiate fusion and simultaneously tag the proteins involved. Ongoing work involves the isolation of cholesterol-enriched membrane fractions to reduce the complexity of the labelled proteome, narrowing the number of candidate proteins.

Hydrophobic Environment of Cysteine Residues in Pig Brain S-100 Proteins

Abstract The effects of metal ion binding to pig brain S-100 proteins on the hydrophobicity of their cysteine (Cys) residues were examined using the fluorescent thiol reagent, N-(1-anilinonaphthyl-4)maleimide (ANM). The hydrophobicity order was Ca2+/S-100 < K+/S-100 < Mg2+/S-100 < S-100 < Zn2+/S-100.

Immunohistochemical localization of sulfhydryl oxidase correlates with disulfide crosslinking in the upper epidermis of rat skin.

Striking morphological changes occur during the terminal stages of epidermal differentiation, such as the loss of major cellular organelles, the aggregation of keratin filaments and the formation of the cornified cell envelope (CCE). The CCE is a highly specialized structure in the cell periphery [1–3], and serves as a physical barrier for the organism and helps to maintain the structural integrity of the upper epidermis. The CCE is rendered insoluble by various post-translational modifications including isopeptide crosslinking and disulfide (S-S) bonding. Isopeptide crosslinking is mainly catalyzed by a membrane-associated transglutaminase specific for the epidermis. During the formation of the CCE this enzyme crosslinks different precursor proteins via N-(gamma-glutamyl)-lysine isopeptide bonds [4–6]. On the other hand, S-S bonding is thought to cause conformational changes of the membrane-associated proteins [7]. We have been interested in S-S crosslinking reactions in the epidermis and have reported in this journal [8, 9] on the clear presence of S-S bonds in human skin as determined using a new fluorescent thiol reagent, N-(7-dimethylamino-4-methyl coumarinyl)-maleimide (DACM; Wako, Tokyo), which specifically binds to thiol groups (SH) causing fluorescence [8, 9]. Using this method we have shown that free -SH groups are distributed in the cytoplasm of all living layers of the epidermis. However, SS bridges are only barely detectable in the living layer, but appear suddenly in the periphery of epidermal cells localized in the transition zone of the living and cornified layers of human skin. Based on this fact, we have suggested that -SH groups suddenly become oxidized to S-S bonds at the junction of the granular and cornified layers [8, 9]. Yukiko Hashimoto · Yasushi Suga · Shouichi Matsuba · Masayuki Mizoguchi · Kenji Takamori · Juergen Seitz · Hideoki Ogawa

Saudi Experience with Classic Homocystinuria

Classic homocystinuria is an autosomal recessive disorder due to cystathionine ss-synthase deficiency. The clinical, radiological and neurophysiological findings of classic homocystinuria diagnosed at King Faisal Specialist Hospital and Research Centre (KFSH&RC) are presented in this report. Twenty-four patients (15 females and 9 males) were referred to KFSH&RC for work-up of mental retardation, seizures, thrombo-embolic episodes and dislocation of the ocular lenses. The common clinical findings included ectopia lentis (20 patients), skeletal system involvement (18 patients), vascular system involvement (9 patients), and mental retardation (all patients to varying degrees). Unusual findings consisted of a patient who developed severe lower gastrointestinal bleeding, a patient with insulin-dependent diabetes mellitus, probably due to vasculopathy, and another having severe bronchiectasis, which may have been due to fibrillin disruption, and required the resection of a lobe of the lung. The parents of 21 patients were first-degree relatives, and 19 patients had one or more family members affected by the same disease. All patients had markedly elevated plasma levels of methionine. Cystathionine synthase activity in the fibroblast was measured in 25% of the patients and was deficient. Only four patients responded to pyridoxine and their methionine level decreased to almost normal range. The aim of this study was to increase the awareness of this disease in the scientific and medical community, in particular in the general pediatrician working in Saudi Arabia who first encounters the clinical manifestations of the disease. Early detection through tandem mass spectrometry of blood spot screening and treatment are important, and may prevent the major complications of this disease.

Cross-Linking andN-(1-Pyrenyl)maleimide Labeling of Cysteine Mutants of Proton-Pumping Pyridine Nucleotide Transhydrogenase ofEscherichia coli

The pyridine nucleotide transhydrogenase of Escherichiacoli is a proton pump composed of two subunits (alpha and beta) organized as an alpha2beta2 tetramer. The enzyme contains seven cysteine residues, five in the alpha-subunit and two in the beta-subunit. The reaction of these residues with the cross-linking agent cupric 1, 10-phenanthrolinate and with the fluorescent thiol reagent N-(1-pyrenyl)maleimide was investigated in mutants in which one or more of these cysteine residues had been mutated to serine or threonine residues. Mutation of alphaCys395 and alphaCys397 prevented disulfide bond formation to give the cross-linked alpha2 dimer. We concluded that the two alpha-subunits of the holoenzyme interface in the region of these two cysteine residues. Pyrenylmaleimide reacted with detergent-washed cytoplasmic membrane vesicles containing high levels of transhydrogenase protein to show characteristic fluorescence emission bands at 378-379, 397-398, and 419-420 nm. At higher ratios of pyrenylmaleimide:transhydrogenase (>5:1) and longer times of reaction, an eximer band at 470 nm was formed. This was attributed to interaction between noncovalently bound molecules of pyrenylmaleimide. The cysteine residues of the beta-subunit (betaCys147 and betaCys260) were covalently modified by pyrenylmaleimide. betaCys147 reacted more strongly than betaCys260 with the fluorophore, and the pyrene derivative of betaCys147 was more accessible to quenching by 5-doxylstearate, suggesting a proximity to the surface of the membrane. Covalent modification of betaCys260 resulted in inhibition of enzyme activity. The inhibition was attributed to the introduction of the bulky pyrene group into the enzyme.

In situ fluorescent visualization of nucleolar organizer region-associated proteins with a thiol reagent.

Nucleolar organizer regions (NORs) are nucleolus-forming rDNA loops associated with argyrophil proteins, the amount of which varies according to the proliferative state of the cell. It has been presumed that the nucleolar protein-related thiol groups may have a role in selective silver staining. We investigated the nuclear thiol distribution with a fluorescent thiol reagent, coumarinyl-phenyl-maleimide (CPM) in human K-562 myeloblast cultures and found that SH group-related fluorescence was brightest in the area of nucleoli, which became highly selective after RNAse digestion. A remarkable co-localization of AgNOR silver reaction and CPM fluorescence was observed, although occupation of the SH groups by CPM did not prevent the silver staining. We applied the stain to dual-parameter flow cytometry in combination with DNA content measurements, which provide further information on nucleolar function and changes in experimental and pathological specimens.

Redox forms of human placenta glutathione transferase.

Human placenta glutathione transferase (EC 2.5.1.18) pi undergoes an oxidative inactivation which leads to the formation of an inactive enzymatic form which is homogeneous in several chromatographic and electrophoretic conditions. This process is pH dependent, and it occurs at appreciable rate in alkaline conditions and in the presence of metal ions. Dithiothreitol treatment completely restores the active form. -SH titration data and electrophoretic studies performed both on the oxidized and reduced forms indicate that one intrachain disulfide is formed, probably between the two faster reacting cysteinyl groups of each subunit. By the use of a specific fluorescent thiol reagent the disulfide forming cysteines have been identified as the 47th and 101th residues. The disulfide formation causes changes in the tertiary structure of this transferase as appears by CD, UV, and fluorometric analyses; evidences are provided that one or both tryptophanyl residues of each subunit together with a number of tyrosyl residues are exposed to a more hydrophilic environment in the oxidized form. Moreover, electrophoretic data indicate that the subunit of the oxidized enzyme has an apparent molecular mass lower than that of the reduced transferase, thereby confirming structural differences between these forms.

Application of new fluorescent thiol reagent to diagnosis of homocystinuria

A new fluorescent thiol reagent, dansylaminophenylmercuric acetate (DAPMA), was applied to the diagnosis of homocystinuria, a disorder which can be associated with vascular disease at an early age. DAPMA was added to urine containing metabisulphite and the resulting fluorescent derivatives were extracted on a cyclohexyl silica column and separated by thin-layer chromatography. 102 coded samples were tested. The derivative of homocysteine was easily identified in samples from 4 children with homocystinuria but was absent from all samples from normal subjects and patients with unrelated disorders. Other thiols (cysteine, acetylcysteine, mercaptolactate, thiosulphate, and thiocyanate) were also identified in urine from healthy fasting subjects.

Synthesis and properties of a maleimide fluorescent thiol reagent derived from a naphthopyranone

AbstractN‐(2‐Carbomethoxy‐9‐methoxy‐3‐oxo‐3H‐naphtho[2,1‐b]pyran‐10‐yl)maleimide 2 was designed and synthesized as a new maleimide fluorescent thiol reagent. The optical properties of 2 were investigated with and without the addition of glutathione, GSH. We have found 2 is twice as sensitive as DACM and is of comparable sensitivity to CPM for detection of thiols. The emission maximum for the GSH adduct of 2 is 513 nm which is at longer wavelength than the GSH adducts of both DACM and CPM.

Chemical modification of rabbit IgG with N-dansylaziridine. Investigation of the properties of dansylated antibodies

To elucidate the effect of antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ), which is sensitive to the changes in the microenvironment, was used for modification of rabbit IgG hinge region cysteine residue. DAZ binds to hinge region Cys 226 as evidenced by the structural analysis. Labelling of IgG with DAZ does not alter its conformation, hydrodynamic behavior, nor its antigen binding properties. Upon antigen binding, the fluorescence intensity of modified IgG increases about 80%. This result indicates that interaction of antibodies with antigen is accompanied by the conformational changes in the IgG hinge region.

Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.

Reactivity of sulfhydryl groups in the brush-border membranes of chick duodena is increased by 1,25-dihydroxycholecalciferol

The total amount and reactivity of SH-groups were determined in isolated duodenal brush-border membranes from rachitic given 1,25-dihydroxyvitamin D-3 (1,25(OH) 2D 3) before isolation of the membranes. I,25(OH) 2D 3 treatment significantly increased the total amount of SH groups (9.7 ± 2.3 vs. 23.9 ± 2.1, P<0.001, n = 6) in brush-border membranes solubilized in 1% sodium dodecyl sulfate. The rate of reaction (reactivity) of membrane-bound SH-groups, determined with the fluorescent thiol reagent N-(7-dimethylamine-4-methylcoumarin-3-yl)maleimide (DACM), was also significantly enhanced by the intravenous injection of various doses (0.005–0.10 μg) of 1,25(OH) 2D 3 into vitamin D-deficient chicks. An increase in reactivity occurred as early as 10 min after dosing of the chicks with 1,25(OH) 2D 3. Fluoresence scanning of the membrane proteins labeled with DACM and separated by polyacrylamide gel electrophoresis revealed three major peaks of fluorescence and a generally higher degree of fluorescent labeling of these and many other proteins in the membranes isolated from the 1,25(OH) 2D 3-treated chicks. The physiological significance of the 1,25(OH) 2D 3-mediated increase in the reactivity and total amount of membrane-bound SH-groups in terms of vitamin-D-dependent epithelial transport and epithelial membrane properties needs to br further explored.

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver.

Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.

Spectral properties of fluorescent derivatives of the oligomycin sensitivity conferring protein and analysis of their interaction with the F1 and F0 sectors of the mitochondrial ATPase complex.

In order to study the kinetics and the nature of the interactions between the oligomycin sensitivity conferring protein (OSCP) and the F0 and F1 sectors of the mitochondrial ATPase complex, fluorescent derivatives of OSCP, which are fully biologically active, have been prepared by reaction of OSCP with the following fluorescent thiol reagents: 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (Mal-ANS), N-(1-pyrenyl)maleimide (Mal-pyrene), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (Mal-coumarin), and fluorescein 5-maleimide (Mal-fluorescein). The preparation of these derivatives was based on the previous finding that the single cysteinyl residue of OSCP, Cys 118, can be covalently modified by alkylating reagents without loss of biological activity [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. For all fluorescent probes used, except Mal-pyrene and Mal-fluorescein, the emission spectra of conjugated OSCP were blue-shifted relative to those of the corresponding mercaptoethanol adducts, indicating that the fluorophores attached to Cys 118 were located in a hydrophobic pocket. These results were consistent with the high quantum yields and the increased fluorescence lifetimes of conjugated OSCP compared to mercaptoethanol adducts in aqueous buffer. They also fit with quenching data obtained with potassium iodide which showed that the fluorophore is shielded from the aqueous medium when it is attached to Cys 118 of OSCP. Especially noticeable was the wide half-width of the OSCP-acrylodan emission peak compared to that of mercaptoethanol-acrylodan.(ABSTRACT TRUNCATED AT 250 WORDS)

Heat-induced thiol-disulfide interchange reaction on the third component of human complement, C3.

The third component of human complement, C3 is composed of two disulfide-bridged polypeptide chains of Mr 120,000 (alpha chain) and Mr 70,000 (beta chain). C3 has a thioester bond that serves as a binding site for targets when C3 is activated. Heat treatment of C3 induces autolytic peptide bond cleavage at the thioester site in the alpha chain as well as rupture of the thioester bond. The alpha chain fragments are linked to each other and beta chain via disulfide bonds. This study, however, documented that prolonged heating gave rise to liberation of several fragments including beta and the larger fragment of alpha chain. Using a fluorescent thiol reagent and [14C]iodoacetamide, we analyzed thiol residues present on each fragment, and elucidated that the thiol residue exposed by rupture of the thioester bond shifts in turn to another fragment resulting in the liberation of the fragments. The results were compatible with those on C4, and suggested that the generated thiol residue induces thiol-disulfide interchange reaction. On heating of plasma, fragments of C3 were not released, while the cleavage of the alpha chain occurred more effectively. The heated C3 (56 degrees C, 15 min) became insusceptible to C3b inactivator (I) and factor H, suggesting that additional conformational change is accompanied with cleavage of the thioester bond.

A conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2+-ATPase upon ATP binding to the catalytic site.

Sarcoplasmic reticulum vesicles were modified with a fluorescent thiol reagent, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. One mol of readily reactive thiols per mol of the Ca2+-ATPase was labeled without a loss of the catalytic activity. The fluorescence of the label increased by 8% upon binding of Ca2+ to the high affinity sites of the enzyme. This fluorescence enhancement probably reflects a conformational change responsible for Ca2+-induced enzyme activation. Upon addition of ATP to the Ca2+-activated enzyme, the fluorescence decreased by 15%. This fluorescence drop and formation of the phosphoenzyme intermediate were determined under the same conditions with a stopped-flow apparatus and a rapid quenching system. The amplitude of the fluorescence drop thus determined was saturated with 3 microM ATP. This shows that the fluorescence drop was caused by ATP binding to the catalytic site. In contrast, the rate of the fluorescence drop was not saturated even with 50 microM ATP. The fluorescence drop coincided with phosphoenzyme formation at 0.5 or 3 microM ATP, but it became much faster than phosphoenzyme formation when the ATP concentration was raised to 100 microM. These results indicate that the ATP-induced fluorescence drop reflects a conformational change in the enzyme.ATP complex. The fluorescence drop was accompanied by a red spectrum shift, which suggests that the label was exposed to a more hydrophilic environment. The electrophoretic analysis of the tryptic digest of the labeled enzyme (10.9 kDa) showed that almost all of the label was located on the 5.2-kDa fragment which includes the carboxyl terminus and the putative ATP-binding domain. The sequencing of the two major labeled peptides, which were isolated from the thermolytic digest of the labeled enzyme, revealed that the labeled site in either of these peptides was Cys674. It seems likely that the label bound to this Cys674 could be involved in the observed fluorescence changes.

Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes.

In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit. Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit. In other work [Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta. As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.

N,N′-dicyclohexylcarbodiimide and 4-chloro-7-nitrobenzofurazan bind to different β subunits of the F 1 ATPase of Escherichia coli

The fluorescent thiol reagent 2-(4′-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS) labels the γ, δ, and one of the three β subunits of the F 1 ATPase from Escherichia coli (ECF 1). This is the same β subunit which incorporates 4-chloro-7-nitrobenzofurazan (Nbf) [H. Stan-Lotter and P. D. Bragg (1986) Eur. J. Biochem. 154, 321–327] . After inactivation of ECF 1 with N,N′-dicyclohexylcarbodiimide (DCCD), IAANS labels in addition to the β, γ, and δ subunits also the α subunit. This suggests a conformational change of ECF 1 upon binding of DCCD. The β subunit which incorporates DCCD does not bind IAANS. Likewise, IAANS-modified ECF 1 does not incorporate DCCD into the same β subunit. It is concluded that DCCD and Nbf bind to different β subunits. Since neither of these reagents binds to that β subunit which can be crosslinked to the ϵ subunit by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, these data show that there is a difference in the chemical reactivity of each of the three β subunits of ECF 1, despite their identical primary structures. This suggests that there is an asymmetry in the F 1 molecule.

Location of the interchain disulfide bonds of the fourth component of human complement (C4): evidence based on the liberation of fragments secondary to thiol-disulfide interchange reactions.

Treatment of human C4 with chemical denaturants and heat produces rapid, autolytic peptide bond cleavage of the alpha-chain. These alpha-chain fragments are linked to the parent C4 molecule through disulfide bonds. On more prolonged heating, however, there is liberation of several peptides, including the beta-chain, the gamma-chain, and a C-terminal alpha-chain fragment. This reaction is inhibited by iodoacetamide. By using a fluorescent thiol reagent and 14C-iodoacetamide, the thiol group present on each peptide was analyzed. The results suggest that the thiol residue exposed by cleavage of the thioester bond induces thiol-disulfide interchange reactions to liberate the peptides. Based on the identification of fragments liberated, the kinetics of their appearance, their sulfhydryl content, and the reported primary structure of human C4, a model of the interchain disulfide bonds is proposed in which the amino terminal portion of the alpha-chain is disulfide-linked to both the beta- and gamma-chains, whereas the carboxyl terminal portion of the alpha-chain is disulfide-linked to only the gamma-chain.