Cleavage of human C5 by trypsin: characterization of the digestion products by gel electrophoresis.

Human C5 is composed of two nonidentical polypeptide chains, alpha and beta (m.w. 130,000 and 80,000, respectively) linked together by disulfide bonds and noncovalent forces. Cleavage of C5 by trypsin fragments with increased anodic mobilities. Limited digestion of C5 by trypsin (substrate to enzyme ratio 10:1 w/w at 37 degrees C for 1 min) resulted in the release of a small terminal alpha-chain peptide (alpha1, m.w. 15,000) probably analogous to C5a, from a large fragment, C5b (m.w. 195,000) composed of an intact beta-chain disulfide linked to an alpha-chain that has a lower m.w. (alpha' 115,000). Further digestion (37 degrees C, 5 min) resulted in cleavage of the alpha-chain at multiple sites with the production of three peptides from the alpha'-chain (alpha2I, 23,500; alpha2II 15,700 and alpha2III 10,200) and a residual fragment, C5c (m.w. 144,000). The alpha1 and alpha2 peptides are not covalently linked to the beta-chain nor to one another. The C5c fragment on the other hand is composed of small peptides of the alpha'c chain (alpha3 14,000; alpha4I 9,000; ALPHA 4II 11,000; alpha 5 23,000 to 30,000) which are linked to the beta-chain and also probably to one another by covalent bonds. Secondary cleavage occurred upon prolonged digestion with trypsin (37 degrees C, 20 min), and this resulted in the progressive erosion of the alpha'c peptides and the conversion of C5c to smaller C5c-like species.     

Cleavage of human fibroblast type I procollagen by mammalian collagenase: demonstration of amino- and carboxy-terminal extension peptides.

Human skin fibroblasts in monolayer culture synthesize and secrete precursor forms of collagen into the culture medium. The type I collagen precursor, the major precursor in the culture medium, was isolated on DEAE cellulose chromatography and subjected to mammalian collagenase cleavage. The amino terminal cleavage fragments had a higher molecular weight than α1^A and α2^A, but did not contain interchain disulfide bonds. The carboxy-terminal cleavage fragments formed high molecular weight aggregates which contained interchain disulfide bonds. These results indicate that human type I procollagen contains noncollagenous amino and carboxy-terminal extension peptides and that all of the interchain disulfide bonds are on the carboxy-terminal portion of the molecule.     

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.     

Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation

Human fibrinogen is a dimer composed of two identical halves. Each dimeric half contains three peptide chains (alpha, beta, and gamma) linked by disulfide bonds. The two half-molecules are joined by three disulfide bonds, one between the two alpha-chains (residue alpha-28) and two between the two gamma-chains (residues gamma-8 and gamma-9). In the absence of any difinitive experimental evidence, it has been presumed that the joined halves were aligned in a parallel orientation similar to the situation found in immunoglobulins. We have now determined that the two gamma-chains--hence, the dimeric halves--are connected in an antiparallel manner. A tryptic peptide containing gamma-chain residues 6-14 was isolated as a disulfide-linked dimer from CNBr-treated fragment E. Synthetic peptides corresponding to this sequence were prepared, from which parallel and antiparallel dimers were constructed. During the syntheses, cysteine thiol groups were protected as p-methoxybenzyl and acetamidomethyl sulfides; the peptides were dimerized by selective deprotection and disulfide bond formation. First, the p-methoxybenzyl groups were removed by liquid hydrogen fluoride and the newly exposed thiols oxidized in the presence of potassium ferricyanide. Then the monocystine compound was converted to the double-cystine product by iodolytic cleavage of the acetamidomethyl group with concomitant disulfide bond formation. This selectivity was used to prepare peptide dimers which modeled both parallel and antiparallel arrangements. The antiparallel-oriented synthetic peptide was indistinguishable from the native tryptic peptide as judged by elution from reverse-phase high-performance liquid chromatography and circular dichroism spectroscopy. The parallel-oriented synthetic peptide differed from the native material by both criteria.     

Development of disulfide peptide mapping and determination of disulfide structure of recombinant human osteoprotegerin chimera produced in Escherichia coli

Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin. The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer. A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule. The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses. Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion. Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay. Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed.     

Disulfide bond assignment of the Ebola virus secreted glycoprotein SGP

The non-structural glycoprotein (SGP) of Ebola virus (EboV) is secreted in large amounts from infected cells as a disulfide-linked homodimer. In this communication, highly purified SGP, derived from Vero E6 cultures infected with the Zaire species of EboV, was used to determine the correct localization of inter- and intrachain disulfide bonds. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis of proteolytic cleavage fragments indicates that all cysteines (six per monomeric unit) form unique disulfide bonds. Monomers of the SGP homodimer are joined in a parallel manner by two intersubunit disulfide bonds formed between paired N-terminal and C-terminal cysteines (C53-C53' and C306-C306'). The remaining cysteines are involved in intrachain disulfide bonding (paired as C108-C135 and C121-C147), which resembles the disulfide bond topology of fibronectin type II domains. The findings presented here provide the foundation for future studies aimed at defining the structural and functional properties of SGP.     

Identification and characterization of disulfide bonds in proteins and peptides from tandem MS data by use of the MassMatrix MS/MS search engine

A new database search algorithm has been developed to identify disulfide-linked peptides in tandem MS data sets. The algorithm is included in the newly developed tandem MS database search program, MassMatrix. The algorithm exploits the probabilistic scoring model in MassMatrix to achieve identification of disulfide bonds in proteins and peptides. Proteins and peptides with disulfide bonds can be identified with high confidence without chemical reduction or other derivatization. The approach was tested on peptide and protein standards with known disulfide bonds. All disulfide bonds in the standard set were identified by MassMatrix. The algorithm was further tested on bovine pancreatic ribonuclease A (RNaseA). The 4 native disulfide bonds in RNaseA were detected by MassMatrix with multiple validated peptide matches for each disulfide bond with high statistical scores. Fifteen nonnative disulfide bonds were also observed in the protein digest under basic conditions (pH = 8.0) due to disulfide bond interchange. After minimizing the disulfide bond interchange (pH = 6.0) during digestion, only one nonnative disulfide bond was observed. The MassMatrix algorithm offers an additional approach for the discovery of disulfide bond from tandem mass spectrometry data.     

Biological regulation through protein disulfide bond cleavage

It is thought that disulfide bonds in secreted proteins are inert because of the oxidizing nature of the extracellular milieu. We have suggested that this is not necessarily the case and that certain secreted proteins contain one or more disulfide bonds that can be cleaved and that this cleavage is central to the protein's function. This review discusses disulfide bond cleavage in the secreted soluble protein, plasmin. Cleavage of plasmin disulfide bond(s) triggers peptide bond cleavage and formation of the tumour angiogenesis inhibitor, angiostatin. Tumour cells secrete phosphoglycerate kinase which facilitates cleavage of the plasmin disulfide bond(s). Phosphoglycerate kinase is not a conventional disulfide bond reductase. We propose that phosphoglycerate kinase facilitates cleavage of a particular plasmin disulfide bond by hydroxide ion, which results in formation of a sulfenic acid and a free thiol. The free thiol is then available to exchange with another nearby disulfide bond resulting in formation of a new disulfide and a new free thiol. The reduced plasmin is then susceptible to discreet proteolysis which results in release of angiostatin.     

Location of the inter-chain disulfide bonds of the third component of human complement

Location of the disulfide bonds connecting three polypeptide chains (alpha 3, 27kd; 2, 43kd; beta, 75kd) of C3c has been investigated by partial reduction with cysteine followed by alkylation with 14C-monoiodoacetic acid. Treatment of C3c with cysteine produced a partially reduced fragment, composed of disulfide-linked beta and alpha 3 chains. A single thiol residue was detected on the alpha 3 chain but not on the beta chain of the fragment, suggesting that the alpha 2 chain in C3c is linked through a single disulfide bond to the alpha 3 chain but not to the beta chain.     

Biological regulation through protein disulfide bond cleavage

Abstract

It is thought that disulfide bonds in secreted proteins are inert because of the oxidizing nature of the extracellular milieu. We have suggested that this is not necessarily the case and that certain secreted proteins contain one or more disulfide bonds that can be cleaved and that this cleavage is central to the protein's function. This review discusses disulfide bond cleavage in the secreted soluble protein, plasmin. Cleavage of plasmin disulfide bond(s) triggers peptide bond cleavage and formation of the tumour angiogenesis inhibitor, angiostatin. Tumour cells secrete phosphoglycerate kinase which facilitates cleavage of the plasmin disulfide bond(s). Phosphoglycerate kinase is not a conventional disulfide bond reductase. We propose that phosphoglycerate kinase facilitates cleavage of a particular plasmin disulfide bond by hydroxide ion, which results in formation of a sulfenic acid and a free thiol. The free thiol is then available to exchange with another nearby disulfide bond resulting in formation of a new disulfide and a new free thiol. The reduced plasmin is then susceptible to discreet proteolysis which results in release of angiostatin.     

A novel methodology for assignment of disulfide bond pairings in proteins.

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.     

Characterization of disulfide bond position in proteins and sequence analysis of cystine-bridged peptides by tandem mass spectrometry.

Tandem mass spectrometry employing high-energy, collisionally activated dissociation (CAD) is shown to be a useful method for sequencing through the cystine bridge of intermolecularly disulfide-bonded peptides. A characteristic triplet of intense fragment ions is observed corresponding to cleavage through and to either side of the disulfide bridge. These fragments define the masses of the linked peptides. Fragments due to peptide chain cleavage are also observed at lower abundance in the product-ion spectra and can be sufficient to sequence both of the disulfide-linked peptides without any prior knowledge of the peptide or protein sequence. Even in cases where the peptide sequence-related product-ion yields are poor, the intensities of the disulfide cleavage ions are usually sufficient to determine the molecular weights of the component cystine-bridged peptides. In this paper we demonstrate that the high-energy CAD tandem MS approach may be used to characterize disulfide-bonded peptides directly in complex enzymatic or chemical digests of native proteins. This obviates the need for individual purification of intermolecularly disulfide-linked peptides prior to analysis. The techniques are illustrated here for synthetic, inter- and intramolecularly disulfide-linked peptides and for human transforming growth factor-alpha (des-Val-Val-TGF-alpha), a compact protein containing 48 residues and three disulfides.     

Disulfide assignments in recombinant mouse and human interleukin 4

The disulfide pairings of mouse and human interleukin 4 (IL-4) proteins have been determined. The purified proteins, synthesized by recombinant DNA technology, are fully active as judged by their ability to stimulate an appropriate biological response in a variety of functional assays. Peptide maps were produced by digesting the proteins with pepsin and separating the resulting fragments by reverse-phase HPLC using linear acetonitrile-TFA gradients. Cystine-containing peptides were identified by determining which reverse-phase peaks showed an altered elution pattern after reduction. These peptides were purified further and defined by composition and sequence analysis. Three sets of disulfide-linked peptides were consistently identified for each protein. For mouse IL-4, the first and fifth, second and fourth, and third and sixth cysteines are joined. The disulfide bonds in human IL-4 are between the first and sixth, second and fourth, and third and fifth cysteines. A large double-loop region within the central three-fifths of each protein is stabilized by these bonds. Sequence analysis of the peptides containing the third and fifth cysteines of human IL-4 also demonstrated that only one of the potential N-glycosylation sites is used by C127 mammary tumor cells. Complete alkylation of mouse IL-4 under mild conditions completely destroyed its biological activity in a hematopoietic precursor cell proliferation assay.     

Local interactions favor the native 8-residue disulfide loop in the oxidation of a fragment corresponding to the sequence Ser-50-Met-79 derived from bovine pancreatic ribonuclease A

A 30-residue peptide was obtained from ribonuclease A by chemical cleavage with cyanogen bromide, subsequent sulfitolysis with concomitant S-sulfonation, and finally enzymatic cleavage withStaphylococcus aureus protease. The peptide was converted to the free thiol form by reductive cleavage of the S-sulfo-protecting groups withd,l-dithiothreitol. This peptide consisted of residues 50–79 of the native sequence of ribonuclease A, with the exception that methionine-79 had been converted to homoserine. Included in this sequence are residues cysteine-65 and cysteine-72, which form a disulfide bond in the native enzyme, as well as cysteine-58. This molecule may form one of three possible intramolecular disulfide bonds upon thiol oxidation, viz. one loop of 15 and 2 of 8 residues each. These isomeric peptides were prepared by oxidation with cystamine, 2-aminoethanethiolation of residual thiols, and fractionation by reverse-phase high-performance liquid chromatography. Disulfide pairings were established by mapping the tryptic fragments and confirming their composition by amino acid analysis. After protracted incubation under oxidizing conditions at 25.0°C andp H 8.0, the 26-member ring incorporating the native disulfide bond between residues 65 and 72 is the dominant product. Assuming that equilibrium is established, we infer that local interactions in the sequence of ribonuclease A significantly stabilize the native 8-residue disulfide loop with respect to the non-native 8-residue loop (G°=–1.1±0.1 kcal mole^–1). The implications of this observation for the oxidative folding of the intact protein are discussed.     

Specific cyanylation and cleavage at cysteine-104 human hemoglobin alpha-chain. A novel approach to the problem of the alpha-chain tryptic core in the study of haemoglobin variants by "fingerprinting" methods.

1. A new approach to the analysis, by "fingerprinting", of the tryptic core region of human haemoglobin alpha-chain is described. 2. The alpha-chain is cyanylated at its single cysteine residue (alpha104) and then split, by exposure to mild alkali, at the N-peptide bond of the resulting beta-thiocyanoalanine residue. 3. The two cleavage fragments, alpha1-103 and alpha104-141, are separated by gel filtration, and the fragment alpha104-141, which contains all the residues of the alpha-chain tryptic core, is digested with pepsin. 4. Preparative "fingerprints" of these peptic peptides yield eight major peptides, which provide complete sequence information for the whole region alpha104-141. 5. The utility of the method is demonstrated by repeating the determination of the substitution in haemoglobin Hopkins-2, a known alpha-chain core variant in which histidine-alpha112 (G19) is replaced by an aspartic acid residue.     

Identification of the disulfide bonds in human plasma protein SP-40,40 (apolipoprotein-J).

SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40,40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(alpha)-Cys107(beta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there was no free sulfhydryl groups in the SP-40,40 molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40,40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40,40.     

Intracellular folding pathway of human chorionic gonadotropin beta subunit.

Few experimental models have been used to investigate how proteins fold inside a cell. Using the formation of disulfide bonds as an index of conformational changes during protein folding, we have developed a unique system to determine the intracellular folding pathway of the beta subunit of human chorionic gonadotropin (hCG). Three folding intermediates of the beta subunit were purified from [35S]cysteine-labeled JAR choriocarcinoma cells by immunoprecipitation and by reverse-phase high performance liquid chromatography (HPLC). To identify unformed disulfide bonds, nonreduced folding intermediates were treated with trypsin to liberate non-disulfide-bound, [35S]cysteine-containing peptides from the disulfide-linked peptides. Released peptides were purified by HPLC and identified by amino acid sequencing. The amount of a peptide that was released indicated the extent of disulfide bond formation involving the cysteine in that peptide. Of the six disulfide bonds in hCG-beta, bonds 34-88 and 38-57 form first. The rate-limiting event of folding involves the formation of the S-S bonds between cysteines 23 and 72 and cysteines 9 and 90. Disulfide bond 93-100, the formation of which appears to be necessary for assembly with the alpha subunit of the hCG heterodimer, forms next. Finally, disulfide bond 26-110 forms after assembly with the alpha subunit, suggesting that completion of folding of the COOH terminus in the beta subunit occurs after assembly with the alpha subunit.     

Natural disulfide bond-disrupted mutants of AVR4 of the tomato pathogen Cladosporium fulvum are sensitive to proteolysis,circumvent Cf-4-mediated resistance,but retain their chitin binding ability

The extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. Here, the four disulfide bonds of AVR4 were identified as Cys-11-41, Cys-21-27, Cys-35-80, and Cys-57-72 by partial reduction with Tris-(2-carboxyethyl)-phosphine hydrochloride, subsequent cyanylation, and base-catalyzed chain cleavage. The resulting peptide fragments were analyzed by mass spectrometry. Sequence homology and the disulfide bond pattern revealed that AVR4 contains an invertebrate (inv) chitin-binding domain (ChBD). Binding of AVR4 to chitin was confirmed experimentally. The three disulfide bonds encompassing the inv ChBD motif are also required for protein stability of AVR4. Independent disruption of each of the three conserved disulfide bonds in AVR4 resulted in a protease-sensitive protein, whereas the fourth disulfide bond appeared not to be required for protein stability. Most strains of C. fulvum virulent on Cf-4 tomato contain Cys to Tyr substitutions in AVR4 involving two (Cys-11-41, Cys-35-80) of the three disulfide bonds present in the inv ChBD motif. These natural Cys to Tyr mutant AVR4 proteins did retain their chitin binding ability and when bound to chitin were less sensitive to proteases. Thus, the widely applied tomato Cf-4 resistance gene is circumvented by C. fulvum by amino acid substitutions affecting two disulfide bonds in AVR4 resulting in the absence of the corresponding AVR4 isoforms in apoplastic fluid. However, these natural isoforms of AVR4 appear to have retained their intrinsic function, i.e. binding to chitin present in the cell wall of C. fulvum, most likely to protect it against the deleterious effects of plant chitinases.     

Polypeptide fragments of the third component of complement in urine of hereditary nephritis patients

Pro-HNP, a urine protein isolated from hereditary nephritis patients, is derived from C3 and resembles the C3c domain. It contains disulfide-linked polypeptides of beta 75, alpha 40, and alpha 28. Plasmin degraded pro-HNP in vitro to HNP, which was also isolated from the urine of patients and which contained disulfide-linked polypeptides of beta 60, alpha 38, and alpha 26, and noncovalently bound polypeptide of beta 17. Amino terminal sequence analyses and amino acid compositions of the seven polypeptides isolated from pro-HNP and HNP show that beta 75 degrades to beta 60 and beta 17 (beta 17 locates at the amino end of beta 75), alpha 40 degrades to alpha 38 (both locate at the carboxyl end of the alpha-chain of C3), and alpha 28 degrades to alpha 26 (both are from the amino end of the alpha'-chain of C3b). These results confirm the enzymatic specificity of plasmin on pro-HNP. In HNP, the half-cystine contents of beta 60, alpha 38, alpha 26, and beta 17 were approximately 3, 12, 3, and 4, respectively. Partial reduction readily released alpha 40 from pro-HNP and alpha 38 from HNP. There were about five intra-chain disulfide bonds in alpha 40 or alpha 38; stepwise reduction of these intra-polypeptide bonds apparently accounted for multiple conformations of alpha 40 or alpha 38.     

Characterization of an apparently lower molecular weight gamma-chain variant in fibrinogen Kyoto I. The replacement of gamma-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site

Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal gamma-chain and a gamma-chain variant (gamma Kyoto I) that has an apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal Mr in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified gamma-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that gamma Asn308 of the gamma-chain variant is replaced by lysine. Purified fragment D1 of fibrinogen Kyoto I also contains two types of D1 gamma-remnants: normal and apparently lower Mr types. Abnormal fragment D1 is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) than normal fragment D1, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-gamma-chain monoclonal antibody. Analysis of peptides released from fragment D1 by plasmin in the presence of EGTA demonstrated the cleavage of the gamma Lys308-Gly309 bond. Fragment D1 of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing gamma Asn308 affects the polymerization of fibrin monomers and that the gamma Asn308----Lys replacement causes a conformational change in the gamma-chain which results in the accelerated cleavage of gamma Lys356-Ala357 and gamma Lys302-Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and Gly309 in the presence of EGTA. During these studies, we found that part of the gamma Lys212-Glu213 bond in fragment D1 is cleaved by plasmin in the presence of EGTA.