Primary structure of the reactive site of human C1-inhibitor

Human C1-inhibitor (C1-Inh) forms an equimolar complex with complement proteinase C1s that is resistant to dissociation by sodium dodecyl sulfate. The formation of this stable complex results in the cleavage of a peptide bond near the carboxyl terminus of the inhibitor and, whereas the bulk of C1-Inh remains covalently bound to the light chain of C1s, the postcomplex inhibitor peptide can be isolated under denaturing conditions. We have sequenced the amino-terminal region of this peptide and deduced that it represents the carboxyl-terminal side of the reactive site of C1-Inh. Limited proteolysis of C1-Inh by Crotalus atrox protease results in an active derivative lacking an amino-terminal peptide of 36 residues. Further proteolysis of this derivative with Pseudomonas aeruginosa elastase inactivates the inhibitor and a peptide is released. The amino-terminal sequence of this peptide overlaps with that of the postcomplex peptide and indicates that the residue imparting primary specificity to the inhibitor is arginine.     

Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement.

1. Digestion of human subcomponent C1q with pepsin at pH4.45 for 20h at 37 degrees C fragmented most of the non-collagen-like amino acid sequences in the molecule to small peptides, whereas the entire regions of collagen-like sequence that comprised 38% by weight of the subcomponent C1q were left intact. 2. The collagen-like fraction of the digest was eluted in the void volume of a Sephadex G-200 column, was was showm to be composed of two major fragments when examined by electrophoresis on polyacrylamide gels run in buffers containing sodium dodecyl sulphate. These fragments were separated on CM-cellulose at pH4.9 in buffers containing 7.5M-urea. 3. Human subcomponent C1q on reduction and alkylation yields equimolar amounnts of three chains, which have been designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. One of the pepsin fragments was shown to be composed of the N-terminal 95 residues of the A chain linked, via residue A4, by a single disulphide bond to a residue in the sequence B2-B6 in the N-terminal 91 residues of the B chain. The second pepsin fragment was shown to be composed of a disulphide-linked dimer of the N-terminal 94 residues of the C chain, the only disulphide bond being located at residue C4.4. The mol. wts. of the unoxidized and oxidized pepsin fragments were estimated from their amino acid compositions to be 20 000 and 18 200 for the A-B and C-C dimers and 11 400, 8800 and 9600 for the collagen-like fragments of the A, B and C chains respectively. Estimation of the molecular weights of the peptic fragments by polyacrylamide-gel electrophoresis run in the presence of sodium dodecyl sulphate gave values that were approx. 50% higher than expected from the amino acid sequence data. This is probably due to the high collagen-like sequence content of these fragments.     

Isolation and characterization of human plasma alpha 1-proteinase inhibitor and a conformational study of its interaction with proteinases.

1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.     

The structure and enzymic activities of the C1r and C1s subcomponents of C1, the first component of human serum complement.

The subcomponents C1r and C1s and their activated forms C-1r and C-1s were each found to have mol.wts. in dissociating solvents of about 83000. The amino acid compositions of each were similar, but there were significant differences in the monosaccharide analyses of subcomponents C1r and C1s, whether activated or not. Subcomponents C1r and C1s have only one polypeptide chain, but subcomponents C-1r and C-1s each contain two peptide chains of approx. mol.wts. 56000 ("a" chain) and 27000 ("b" chain). The amino acid analyses of the "a" chains from each activated subcomponent are similar, as are those of the "b" chains. The N-terminal amino acid sequence of 29 residues of the C-1s "a" chain was determined, but the C-1r "a" chain has blocked N-terminal amino acid. The 20 N-terminal residues of both "b" chains are similar, but not identical, and both show obvious homology with other serine proteinases. The difference in polysaccharide content of the subcomponents C-1r and C-1s is most marked in the 'b' chains. When tested on synthetic amino acid esters, subcomponent C-1r hydrolysed both lysine and tyrosine ester bonds, but subcomponent C-1r did not hydrolyse any amino acid esters tested nor any protein substrate except subcomponent C1s. The lysine esterase activity of subcomponent C1s provides a rapid and sensitive assay of the subcomponent.     

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.     

Clostripain-catalyzed re-formation of a peptide bond in a cytochrome C fragment complex

Enzymatically-catalyzed condensation of cytochrome c fragments, ferrous heme fragment (1-38) and apofragment (39-104), has allowed the back-conversion of cytochrome c complex to native cytochrome c. The conversion was accomplished in 90% (v/v) glycerol, a solvent which has been shown to decrease the ionization of the terminal alpha-carboxyl group liberated during hydrolysis of a peptide bond. The effect on the pK is probably the main reason the thermodynamic obstacle to re-synthesis is minimized. A 30% conversion to cytochrome c was obtained. The cytochrome c product was distinguished from the non-covalent complex and separated fragments by molecular weight analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis, by elution from Sephadex G-50 and sulfopropyl-Sephadex in the presence of denaturant, by amino acid analysis of the product purified under complex-dissociation conditions, and by spectral analysis of the absorption bands of the heme. This method provides an opportunity to study the covalent rather than the complex form of cytochrome c analogs.     

Fluid-phase interaction of C1 inhibitor (C1 Inh) and the subcomponents C1r and C1s of the first component of complement, C1.

Interactions between proenzymic or activated complement subcomponents of C1 and C1 Inh (C1 inhibitor) were analysed by sucrose-density-gradient ultracentrifugation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The interaction of C1 Inh with dimeric C1r in the presence of EDTA resulted into two bimolecular complexes accounting for a disruption of C1r. The interaction of C1 Inh with the Ca2+-dependent C1r2-C1s2 complex (8.8 S) led to an 8.5 S inhibited C1r-C1s-C1 Inh complex (1:1:2), indicating a disruption of C1r2 and of C1s2 on C1 Inh binding. The 8.5 S inhibited complex was stable in the presence of EDTA; it was also formed from a mixture of C1r, C1s and C1 Inh in the presence of EDTA or from bimolecular complexes of C1r-C1 Inh and C1s-C1 Inh. C1r II, a modified C1r molecule, deprived of a Ca2+-binding site after autoproteolysis, did not lead to an inhibited tetrameric complex on incubation with C1s and C1 Inh. These findings suggest that, when C1 Inh binds to C1r2-C1s2 complex, the intermonomer links inside C1r2 or C1s2 are weakened, whereas the non-covalent Ca2+-independent interaction between C1r2 and C1s2 is strengthened. The nature of the proteinase-C1 Inh link was investigated. Hydroxylamine (1M) was able to dissociate the complexes partially (pH 7.5) or totally (pH 9.0) when the incubation was performed in denaturing conditions. An ester link between a serine residue at the active site of C1r or C1s and C1 Inh is postulated.     

The covalent nature of the human antithrombin III--thrombin bond.

1. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III--thrombin complex, the inhibitor is cleaved at an arginine--X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).     

Characterization of recombinant C1 inhibitor P1 variants.

Twelve human C1 inhibitor P1 variants were constructed by site-directed mutagenesis of the codon for arginine 444 and were expressed in COS-1 cells to analyze the functional properties. The ability to bind to target proteases, as well as potential substrate-like behavior, was investigated with radioimmunoassays. The P1-Lys variant retained binding capacity toward C1s, plasmin, and kallikrein. In addition, complex formation with C1s was detected for P1-Asn and P1-His. All other P1 substitutions resulted in C1 inhibitor variants that neither complexed with nor were inactivated by C1s, kallikrein, beta-factor XIIa, or plasmin. Electrophoretic studies confirmed that P1-Lys and P1-His can form sodium dodecyl sulfate-resistant complexes with C1s. In contrast, the C1s-P1-Asn complex dissociated upon addition of sodium dodecyl sulfate. Kinetic experiments by the method of progress curves generated association rate constants (kon) with C1s of 4.2 x 10(4) M-1 s-1 for recombinant wild-type C1 inhibitor and 1.7 x 10(4) M-1 s-1 for P1-Lys. For P1-Asn and P1-His, kon was decreased approximately 100-fold. The results from inhibition experiments were compatible with a model of reversible inhibition, although the observed dissociation rate for wild-type C1 inhibitor is too low (1-2 x 10(-6) s-1) to be physiologically relevant. The overall inhibition constant (Ki) was estimated to be 0.03 nM. With P1-Asn, reversible inhibition could be demonstrated directly upon dilution of preformed complexes; the observed dissociation rate constant was 3.2 x 10(-4) s-1; and Ki increased to approximately 380 nM. These findings are discussed in relation to inhibitor specificity and inhibition mechanism.     

Interaction of human plasma kallikrein and its light chain with C1 inhibitor

The light chain of human plasma kallikrein contains the enzymatic active site. The inactivation of kallikrein and of its isolated light chain by C1 inhibitor was investigated to assess the functional contributions of the heavy-chain region of kallikrein and of high molecular weight kininogen to this reaction. The second-order rate constants for the inactivation of kallikrein or its light chain were respectively 2.7 X 10(6) and 4.0 X 10(6) M -1 min -1. High molecular weight kininogen did not influence the rate of kallikrein inactivation. The nature of the complexes formed between kallikrein or its light chain and C1 inhibitor was studied by using sodium dodecyl sulfate (SDS) gradient polyacrylamide slab gel electrophoresis. Kallikrein as well as its light chain combined with C1 inhibitor to form stable stoichiometric complexes that were not dissociated by SDS and that exhibited apparent molecular weights (Mr's) of 185 000 and 135 000, respectively, on nonreduced SDS gels. Reduction of the kallikrein-C1 inhibitor complex gave a band at Mr 135 000 that comigrated with the complex seen for the light chain-C1 inhibitor complex. During the inactivation of both kallikrein and its light chain, a Mr 94 000 fragment of C1 inhibitor was formed which was unable to inactivate or bind kallikrein or its light chain. Kallikrein inactivated by diisopropyl phosphofluoridate did not form SDS-stable complexes with C1 inhibitor. These results demonstrate that the functional binding site for C1 inhibitor is localized in the light chain of kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human complement component C4. Structural studies on the fragments derived from C4b by cleavage with C3b inactivator.

1. One of the activation products of C4, C4b, was prepared, and the reactive thiol group on the alpha'-chain was radioactively labelled with iodo[2-14C]acetic acid. The alpha'-chain was isolated and the N-terminal amino acid sequence of the first 13 residues was determined. 2. C4b was cleaved by C3bINA in the presence of C4b-binding protein and C4d and C4c isolated. The radioactive label and therefore the reactive thiol group were located to C4d. 3. C4c was reduced and alkylated and the two alpha'-chain fragments of C4c were separated. 3. The molecular weights, amino acid analyses and carbohydrate content of the three alpha'-chain fragments were determined. C4d has a mol.wt. of 44500 and a carbohydrate content of 6%. The two alpha'-chain fragments of C4c have mol.wts. of 25000 (alpha 3) and 12000 (alpha 4) and carbohydrate contents of 10 and 22% respectively. 4. The N-terminal amino acid sequences of C4d, the alpha 3 and the alpha 4 fragments were determined for 18, 24 and 11 residues respectively and, by comparison with the N-terminal sequence of the C4b alpha'-chain, the 25000-mol.wt. fragment (alpha 3) was shown to be derived from the N-terminal part of the alpha'-chain. 5. C-Terminal analyses were done on the alpha'-chain and its three fragments. Arginine was found to be the C-terminal residue of C4d and of the alpha 3 fragment. The C-terminal residue of the alpha'-chain and of the alpha 4 fragment could not be identified. The order of the three fragments of the alpha'-chain is therefore: alpha 3(25000)--C4d(44500)--alpha 4(12000). The specificity of C3bINA is for an Arg--Xaa peptide bond.     

The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000+/-2000 in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca(2+) and is adsorbed to BaSO(4). The pK(a) of its BaSO(4)-binding group(s) is 3.1-3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO(4) and, since the ability of the whole protein to bind to BaSO(4) is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid.     

Analysis of the conformation and stability of human bronchial lysozyme by circular dichroism.

1. Human bronchial lysozyme was isolated from nonpurulent secretions and studied by circular dichroism (CD) spectroscopy for its conformational properties. 2. The two negative bands at 208 and 222 nm indicated that the peptide chain adopted an alpha-helical structure in physiological conditions. 3. The molecule was stable at pH 1.0 but not at pH 12.0. 4. Increasing ionic strength by adding NaCl up to 1 M did not change the CD spectra. 5. Complete unfolding of the molecule by guanidinium chloride was obtained only at the concentration of 6 M. 6. Bronchial lysozyme was also denatured by sodium dodecyl sulphate. 7. The molecule was stable when mild reduction was performed at 37 degrees C for 30 min but was completely unfolded after heating at 100 degrees C for 3 min.     

A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus)

An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.     

The yeast aminopeptidase Y

A metal-dependent aminopeptidase (EC 3.4.11.-), designated APase Y, has been purified to homogeneity by conventional methods. The enzyme is composed of a single polypeptide chain with molecular mass of 102 kilodaltons, estimated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis, with a blocked N-terminal amino acid. It possesses neither endopeptidase nor carboxypeptidase activity and is strongly inhibited by metal-chelating agents, Zn2+, and the protein inhibitor from Neurospora crassa. APase Y is insensitive to Cl anions, S--S reducing reagents, serine protease inhibitors, and the peptidase inhibitor benzamidine. Co2+, Hg2+, and p-chloromercuribenzoate can activate the enzyme up to 22, 20, and 55%, respectively. The holoenzyme is resistant to yeast endopeptidases A, B, and Y, whereas the apoenzyme (obtained after treatment with chelators) is susceptible to the serine endopeptidases B and Y. The enzyme catalyzes hydrolysis of most L peptides possessing free alpha-amino (or imino) group by stepwise removal of N-terminal residue. Peptides with L-leucine at the N terminus are cleaved preferentially. The enzyme is unable to catalyze hydrolysis of X--Pro type peptide bonds, and inefficiently hydrolyzes bonds between Asp--X and Glu--X. L-leucine p-nitroanilide hydrolyzes optimally at pH 8.2 with a Km value of 1 mM. The purified enzyme is stable during storage in 0.05 M phosphate buffer, pH 6.7, containing 40-50% glycerol, at -20 degrees C.     

Isolation and characterization of an inhibitory subunit of the Mg2+--Ca2+-ATPase of Escherichia coli.

1. Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that the ATPase activity both in the membrane-bound and the solubilized form is partly masked. 2. A protein, inhibiting the ATPase activity of Escherichia coli, can be isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified ATPase. The inhibitor was identified with the smallest of the subunits of E. coli ATPase. 3. The molecular weight of the ATPase inhibitor is about 10,000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and deduced from the amino acid composition. 4. The inhibitory action is independent of pH, ionic strength or the presence of Mg2+ or ATP. 5. The ATPase inhibitor is heat-stable, insensitive to urea but very sensitive to trypsin degradation. 6. The Escherichia coli ATPase inhibitor does not inhibit the mitochondrial or the chloroplast ATPase.     

Modification of the isoinhibitors of human serum alpha 1-proteinase inhibitor (alpha 1-antitrypsin) by pancreatic proteases

Due to the action of a serum protease, the two most cathodal isoinhibitors of the alpha 1-proteinase inhibitor (alpha 1-PI) are cleaved at the Gly5-Asp6 bond and lack two negative charges. In spite of this, these can bind trypsin and chymotrypsin, showing that the N-terminal pentapeptide is not indispensable for inhibition function. Pancreatic proteases also cleave a bond near the N-terminus in alpha 1-PI, resulting in a loss of two negative charges and a corresponding cathodal shift in the electrofocusing behavior of the isoinhibitors. Trypsin cleaves isoinhibitors near the N-terminus at a large inhibitor excess and unless an additional cleavage takes place, at least two of the new isoinhibitors remain active. An additional cleavage(s), most likely at a distance of 30-40 residues from the C-terminus results in a corresponding decrease of the molecular mass and a loss of inhibition function. Although the C-terminal cleavage peptide does separate from the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it remains associated with it under conditions of polyacrylamide gel isoelectric focusing. Chymotrypsin also cleaved alpha 1-PI near the N-terminus but this could be observed only at protease excess and the modified isoinhibitors did not form complexes with chymotrypsin. The molecular polymorphism of alpha 1-PI is partly explained by the absence of the N-terminal pentapeptide from some of the isoinhibitors.     

Chemical and physical properties of the human urinary glycoprotein with gastric antisecretory activity.

The chemical and physical properties of the high-molecular-weight glycoprotein (SO20, w = 8S; Ve=Vo on Sephadex G-200) with gastric antisecretory activity extracted from the urine of pregnant women were studied. Gel filtration in the presence of sodium dodecyl sulphate and sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis indicated subunit mol.wts. of 16 000 +/- 1500 and 13 000 +/- 1000 respectively. Reaggregation of the subunits and partial recovery of the biological activity were observed on removal of the detergent. The partial C-terminal sequence was found to be Phe-Tyr-Leu-Val-OH, whereas glycine appears to be the N-terminal amino acid. The carbohydrate composition was examined; all galactosamine was found to be O-glycosidically linked to the polypeptide chain.     

Expression of functional human C1 inhibitor in COS cells

Full length human C1 inhibitor cDNA was cloned into a vector suitable for transient expression in COS-1 cells. Transfected COS cells secreted an immunoreactive protein of Mr approximately 110,000 that appeared to be functionally equivalent to the plasma-derived protein as established by the following criteria: 1) ability to form sodium dodecyl sulfate-stable complexes with C1s, factor XIIa, and kallikrein; 2) inhibition of C1s-mediated C4 consumption; and 3) susceptibility to inactivation by the nontarget proteinase elastase. Quantitation of secreted recombinant C1 inhibitor by radioimmunoassay indicated that 72 h after transfection the level was approximately 2.2 micrograms/ml. Treatment of transfected cells with tunicamycin resulted in secretion of a protein of Mr approximately 90,000 that was also capable of complex formation with C1s.     

On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides.

A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII by thrombin must be accompanied by a limited proteolysis of the arginyl-glycyl bond located in the N-terminal region of the a chain, liberating the "Activation peptide." The possibility of activating Factor XII with other porteinases was examined using Factor Xa [EC 3.4.21.6], Factor XIIa, kallikreins [EC 3.4.21.8], urokinase [EC 3.4.99.26], trypsin [EC 3.4.21.4], ficin [EC 3.4.22.3], papain [EC 3.4.22.2], and bromelain [EC 3.4.22.4]. Among these enzymes, only bromelain and trypsin showed clear activating effects.