The integrity of the cysteine 186-cysteine 209 bond of the second disulfide loop of tissue factor is required for binding of factor VII

The structural basis of function of tissue factor (TF), the cell surface receptor and cofactor for the serine protease factor VIIa, cannot be inferred from the primary sequence. The functional significance of the two disulfide bonded loops in the surface domain of TF has been analyzed using site-directed mutagenesis to selectively preclude covalent stabilization of these loops by pairwise substitution of serine residues for cysteines. Mutant TF lacking either the amino (TFS49S57) or carboxyl (TFS186S209) disulfide bond were expressed on the surface of cells consistent with proper processing. Each reacted with a panel of monoclonal antibodies further suggesting proper global folding of the mutant proteins. TFS186S209 exhibited a selective decrease in reactivity with an antibody directed against one epitope locus in the carboxyl aspect of the surface domain of TF. Whereas TFS49S57 was functionally comparable to the wild type protein, TFS186S209 was functionally 30-40-fold less effective, and the affinity of factor VIIa binding to this mutant was indirectly estimated to be diminished 20-fold. These data suggest that the Cys186-Cys209 disulfide bond is required to maintain conformation and implicate the disulfide loop or adjacent structures in the carboxyl half of the surface domain of TF in receptor function.     

Kinetics of disulfide bond reduction in alpha-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond

Kinetics of disulfide reduction in alpha-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25 degrees C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and -120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by bimolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide alpha-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon alpha-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.     

Functional implication of disulfide bond, Cys250 -Cys283, in bovine chymosin.

The reduction, carboxymethylation and mercuration of disulfide bond, Cys250-Cys283, located on the surface of bovine chymosin molecule resulted in the loss of about 25% of enzyme activity, suggesting that Cys250-Cys283 is not intimately involved in catalytic mechanism. Cys250 and Cys283 were substituted with Asp. and Ser. by site- directed mutagenesis of the structural gene coding for bovine prochymosin B. All three mutants (C250D/C283S, C250D, C283S) failed to be activated to chymosin in acid, indicating that Cys250-Cys283 might have some contribution to the correct refolding of the unfolded prochymosin.     

Functional implications of disulfide bond, Cys206-Cys210, in recombinant prochymosin (chymosin)

Prochymosin (chymosin) contains three disulfide bonds: Cys45-Cys50, Cys206-Cys210, and Cys250-Cys283. We have demonstrated that Cys250-Cys283 is indispensable for correct refolding of prochymosin, whereas Cys45-Cys50 is dispensable but has some contribution to the stability and substrate specificity of the enzyme. Here, we report the results about the functions of Cys206-Cys210 by site-directed mutagenesis studies. In a glutathione redox system C206A/C210A mutant exhibited oxidative refolding kinetics and efficiency ( approximately 40% reactivation) similar to those of the wild-type prochymosin, indicating that Cys206-Cys210 is also dispensable for refolding. However, C206S/C210S and single-site mutants (C210A, C210S, and C206A) showed only about 3 and 0-0.4% reactivation, respectively. This is quite different from the Cys45-Cys50 deficient mutants (C45A, C50A, C45A/C50A, C45D, C50S, C45D/C50S, C45A/C50S), which have comparable refolding efficiencies, implying that the substituents at position 206 and 210 play more important role in determining correct refolding than those at position 45 and 50. Urea-induced denaturation and fluorescence quenching studies indicated that the prochymosin mutants C206A/C210A and C206S/C210S were 2.1 and 4.8 kJ/mol less stable than prochymosin and some tryptophan residue in the mutated molecules was less exposed. However, the wild-type and mutant prochymosins shared similar far-UV CD and fluorescence emission spectra and similar specific potential activity, suggesting that the overall conformation was maintained after mutation. Activity assay and kinetic analysis revealed that mutation did not change the specific milk-clotting activity significantly but resulted in an increase in K(m) and k(cat) toward a hexapeptide substrate. On the basis of the above-mentioned perturbance of tryptophanyl microenvironment and the three-dimensional structure of chymosin, we proposed that deletion of Cys206-Cys210 may induce a propagated conformational change, resulting in a perturbance of the local conformation around active-site cleft and in turn, an alteration of the substrate specificity.     

Function of disulflde bond Cys45-Cys50 of prochymosin

The conditions (temperature, time, pH) for solubilizing inclusion bodies of prochymosin mutant, Cys45Asp/Cys50Ser, are identical with those for the wild type. Moreover, they have similar oxidative refolding behavior. Under the same renaturation conditions both of them can undergo correct refolding leading to the formation of activable molecules. This is quite different from the mutant with deletion of Cys250-Cys283, indicating that Cys45-Cys50 contributes less to the correct refolding of prochymosin than Cys250- Cys283. However, deletion of Cys45-Cys50 results in a remarkable decrease of the thermostability of pseudochymosin, suggesting that this disulfide bond plays an important role in stabilizing enzyme conformation. The proteolytic (P) and milk-dotting (C) activities of the mutant of pseudochymosin, Cys45Asp/Cys50Ser, are lower than those of its wild counterpart. The C/P ratio of the former is onefold higher than that of the latter.     

Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.     

The role of short-range Cys171-Cys178 disulfide bond in maintaining cutinase active site integrity: A molecular dynamics simulation

Understanding structural determinants in enzyme active site integrity can provide a good knowledge to design efficient novel catalytic machineries. Fusarium solani pisi cutinase with classic triad Ser-His-Asp is a promising enzyme to scrutinize these structural determinants. We performed two MD simulations: one, with the native structure, and the other with the broken Cys171-Cys178 disulfide bond. This disulfide bond stabilizes a turn in active site on which catalytic Asp175 is located. Functionally important H-bonds and atomic fluctuations in catalytic pocket have been changed. We proposed that this disulfide bond within active site can be considered as an important determinant of cutinase active site structural integrity.     

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347

NEMO is an essential regulatory component of the IκB kinase (IKK) complex, which controls activation of the NF-κB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H₂O₂) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H₂O₂ decreases TNFα-induced IKK activity in NEMO-reconstituted cells, and that TNFα has a diminished ability to activate NF-κB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.     

Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions

Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibalpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys(209)-Cys(248) disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on (125)I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibalpha. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibalpha, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibalpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.     

Functional and structural roles of the Cys14-Cys38 disulfide of bovine pancreatic trypsin inhibitor

The disulfide bond between Cys14 and Cys38 of bovine pancreatic trypsin inhibitor lies on the surface of the inhibitor and forms part of the protease-binding region. The functional properties of three variants lacking this disulfide, with one or both of the Cys residues replaced with Ser, were examined, and X-ray crystal structures of the complexes with bovine trypsin were determined and refined to the 1.58-Å resolution limit. The crystal structure of the complex formed with the mutant with both Cys residues replaced was nearly identical with that of the complex containing the wild-type protein, with the Ser oxygen atoms positioned to replace the disulfide bond with a hydrogen bond. The two structures of the complexes with single replacements displayed small local perturbations with alternate conformations of the Ser side chains. Despite the absence of the disulfide bond, the crystallographic temperature factors show no evidence of increased flexibility in the complexes with the mutant inhibitors. All three of the variants were cleaved by trypsin more rapidly than the wild-type inhibitor, by as much as 10,000-fold, indicating that the covalent constraint normally imposed by the disulfide contributes to the remarkable resistance to hydrolysis displayed by the wild-type protein. The rates of hydrolysis display an unusual dependence on pH over the range of 3.5-8.0, decreasing at the more alkaline values, as compared with the increased hydrolysis rates for normal substrates under these conditions. These observations can be accounted for by a model for inhibition in which an acyl-enzyme intermediate forms at a significant rate but is rapidly converted back to the enzyme-inhibitor complex by nucleophilic attack by the newly created amino group. The model suggests that a lack of flexibility in the acyl-enzyme intermediate, rather than the enzyme-inhibitor complex, may be a key factor in the ability of bovine pancreatic trypsin inhibitor and similar inhibitors to resist hydrolysis.     

Evidence for activation of tissue factor by an allosteric disulfide bond

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.     

Role of the Cys18-Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.     

Redox properties of protein disulfide bond in oxidized thioredoxin and lysozyme: a pulse radiolysis study

We have studied the one-electron reduction of oxidized Chlamydomonas reinhardtii thioredoxin and compared it to that of hen egg white lysozyme, using CO(2)(*) (-) free radicals as reductants. This comparison shows that the thioredoxin disulfide/thiol redox couple has different properties than that of lysozyme: the disulfide radical pK(a) is much lower (around 5 for small disulfides, 4.62 for lysozyme, <3 for thioredoxin). To get a better understanding of the modulation of the thioredoxin redox properties we have constructed the mutants W35A and D30A. Their reduction by pulse radiolysis indicates that W35 strongly controls both the disulfide radical acidity (the pK(a) in W35A is equal to ca. 4), and the thiol reactivity. Asp30 is also involved in the control of proton transfer to the disulfide free radical. In addition, its removal seems to increase the reduction potential of the thioredoxin thiyl/thiol couple. Overall, the reduction properties of thioredoxin confirm its nature as a unique reductant.     

Relative spatial position of a snake neurotoxin and the reduced disulfide bond alpha (Cys192-Cys193) at the alpha gamma interface of the nicotinic acetylcholine receptor

We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snake neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) located at the alphagamma interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 A. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine alpha(192-193) by gel densitometry of Coomassie Blue-stained gels. A nearly quantitative coupling was observed between alphaCys(192) and/or alphaCys(193) and all probes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis alpha-neurotoxin. These data sufficed to locate the reactive thiolate in a "croissant-shaped" volume comprised between the first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Altogether, the data suggest that alphaCys(192) and/or alphaCys(193), at the alphagamma interface of a muscular-type acetylcholine receptor, is (are) located in a volume located between 11.5 and 15.5 A from the alpha-carbons at positions 10 and 33 of the toxin, under the tip of the toxin first loop and close to the second one.     

Attempt to map the receptor binding sites of human follicle-stimulating hormone using disulfide peptides of its beta-subunit indicates major involvement of the regions around disulfide bonds Cys28-Cys82 and Cys32-Cys84 in receptor binding of the hormone.

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone secreted by the anterior pituitary. It plays a very important role in folliculogenesis in females and is responsible for spermatogenesis in males. The alpha-subunit which is common within a species and the beta-subunit which is hormone-specific are held together by noncovalent association. This association is very essential for the biological activity of the hormone. Each of these subunits are highly cross-linked by disulfide bonds which appear to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This study was initiated to delineate the role of the disulfide bonds of hFSH beta in receptor binding of the hormone. Five intermolecular and one intramolecular disulfide peptides corresponding to the disulfide bonds found in hFSH beta were synthesized and screened along with their linear counterparts, for their ability to competitively inhibit the radiolabelled [125I]hFSH from binding to the FSH receptor containing membranes from the testis of immature rats. The disulfide peptides Cys28-Cys82 and Cys32-Cys84 were found to be the most potent in inhibiting radiolabelled hFSH from binding to its receptor. The results suggest the involvement of the regions around disulfide bonds Cys28-Cys82 and Cys32-Cys84 in receptor binding of the hormone. The studies also suggest the involvement of beta L2 and beta L3 loop regions in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hFSH.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

Redox properties of the disulfide bond of human Cu,Zn superoxide dismutase and the effects of human glutaredoxin 1

Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [H(C)/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting H(C)/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis.     

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ?i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its beta-subunit: possible involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding of the hormone.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.