Purification of Cathepsin B from Squid Liver

Cathepsin B [EC 3. 4. 22. 1] from the liver of squid, Dorytheuthis bleekri, was purified by the following steps : homogenization, ammonium sulfate fractionation, CM-cellulose column chromatography and rechromatography, Sephadex G–75 column chromatography and iso-electric focusing. The purified enzyme appears to be homogeneous by polyacrylamide gel electrophoresis at both pH 4.3 and pH 9.5 and has an isoelectric point of 6.8. The molecular weight of the enzyme was determined to be 13,600 by Sephadex G–75 column chromatography. The optimum pH for hydrolysis of α-N-benzoyl-l-argininamide (BAA), α-N-benzoyl-dl-arginine-2-naphthylamide (BANA) was 4.5, and it was 4.2~4.7 for N, N′-dimethyl hemoglobin. The value of Km for the hydrolysis of BANA was estimated to be 1.25 mm.     

Specificity and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L) Dc)

The specificity of the winged bean chymotrypsin inhibitor is restricted to the chymotrypsins (EC 3.4.21.1 and EC 3.4.21.2). Trypsins (EC 3.4.21.4), elastase (EC 3.4.21.11), subtilisins (EC 3.4.21.14), proteinase K (EC 3.4.21.14) and Pronase (EC 3.4.24.4) are not inhibited. The inhibitor reacts with two molecules of chymotrypsin to form a stable complex (Mr approx. 70 0000) which was isolated by gel filtration on Sephadex G-100. When mixed with substrate, the interaction of the inhibitor with alpha-chymotrypsin is characterized by substrate-induced dissociation of the complex. In contrast, the interaction with chymotrypsin B is quantitative with no substrate-induced dissociation. The inhibitor reacts with alpha-chymotrypsin to form a 1 : 2 molar complex at all ratios of [I]/[E]; however, the interaction with chymotrypsin B is characterized by the formation of initially of a 1 : 1 molar complex at [I] greater than [E] followed by the formation of the 1 : 2 molar complex at [I] less than 2[E]; an intermediate species of Mr approx. 48 000 was demonstrated by gel filtration on Sephadex G-100. The inhibitor is stable over the pH range 2.0-11.5 and to heating up to 70 degrees C at pH 4.1 and 8.0, and up to 90 degrees C at pH 3.0. The inhibitor resists denaturation in 8.0 M urea at pH 8.0 and 4.0, is stable in 0.12 M beta-mercaptoethanol at pH 8.0; however, reduction in 8.0 M urea results in a loss of inhibitory activity. The inhibitor resists digestion with pepsin at pH 2.0, being only slowly degraded over a period of 7 days with an equimolar amount of pepsin.     

Inhibition of Growth of L5178Y Leukemia Cells by a Fungal Folate-deaminating Enzyme

Reactive sites of adzuki bean proteinase inhibitor II were determined by limited hydrolyses with catalytic amounts of trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1] at pH 3.0. Treatment of the trypsin-modified inhibitor with carboxypeptidase B [EC 3.4.12.3] released lysine from the inhibitor and led to complete loss of the activity for trypsin, virtually, without affecting the chymotrypsin-inhibitory activity. Limited hydrolysis with chymotrypsin resulted in a selective cleavage of a single tyrosyi peptide bond in the inhibitor, and treatment of this modified inhibitor with carboxypeptidase A [EC 3.4.12.2] abolished the chymotrypsininhibitory activity, having no effect on the trypsin-inhibitory activity. After reduction and S-carboxymethylation, the trypsin- and the chymotrypsin-modified inhibitors both could be separated into two components by gel-filtration on Sephadex G–50 and DEAE-cellulose chromatography. Amino acid and end group analyses of these components indicated that the reactive sites of inhibitor II are the Lys²⁷-Ser²⁸ bond against trypsin and the Tyr⁵⁴-Ser⁵⁵ against chymotrypsin.

Chemical modification of inhibitor II with cyanogen bromide had a fatal effect on the inhibitory activity against trypsin but no effect against chymotrypsin.     

Carbohydrate metabolism in one- and two-year-old spruce needles, and stem carbohydrates from three months before until three months after bud break

Starch and sucrose metabolism of one- and two-year-old needles of Norway spruce (Picea abies [L.] Karst., about 30 years old) was investigated from three months before until three months after bud break at a natural site. We distinguish different metabolic states according to the extractable activities of enzymes (α-amylase [EC 3.2.1.1], ADP-glucose pyrophosphorylase [AGP, EC 2.7.7.27], D-enzyme [EC 2.4.1.25], starch phosphorylase [STP. EC 2.4.1.1]), sucrose phosphate synthase [SPS, EC 2.4.1.14], sucrose syntbase [SS, EC 2.4.1.13]. acid invertase [AI, EC 3.2.1.261) and pool sizes of related metabolites (starch, glucose, fructose, sucrose, raffinose, stachyose, fructose 6-phosphate [F6P], glucose 6-phosphate [G6P], fructose 2,6-bisphosphate [F26BP], and inorganic phosphate [P₁]). The period ending with bud break was characterized by high rates of net photosynthesis, a pronounced decrease in the amount of soluble sugars, and a steep rise in starch (from the detection limit to approximately 600 nmol glycosyl units [mg dry weight]_-1). In parallel, the extractable activity of AGP increased, while D-enzyme was on a relative high level when compared with the period after bud break. With respect to sucrose metabolism, F26BP, an inhibitor of sucrose synthesis, decreased from 1 to 0.4 pmol (mg dry weight)_-1. This was complemented by SPS activity, which was due to both increased protein levels shown by immunoblotting and activation under metabolite control (high levels of G6P and a low P_i/G6P ratio). This indicates a high capacity of synthesis of starch and sucrose in the period before bud break. These observations are in accordance with estimates of photosynthetic carbon gain, which indicate that in early spring large amounts of carbon from current photosynthesis are exported out of the needles. In addition, the content of nonstructural carbohydrates (expressed as hexoses) increased in the bark of the stem. This could also be a consequence of an enhanced carbon export from the needles. After the onset of bud break, starch concentration decreased in all tissues under investigation. In contrast, the level of total nonstructural carbohydrates in the outermost sapwood nearly doubled from bud break until the end of sampling. In the needles, net photosynthesis was reduced by about 75% and a decrease in SPS activity and protein level were found together with lower G6P concentration, and an increased P_i/G6P ratio. These results suggest that during that period sucrose synthesis was reduced in the older needles. In addition, under conditions of reduced photosynthesis, carbon demand of current year needles was in part ensured by the mobilization of starch in the older needles. Taken together our data show that before bud break carbon metabolism of mature leaves is related with the sink demands of storage organs. After bud break the accumulated assimilate pools in needles and stem, mainly the bark, are mobilized and support carbon supply to new tissues.     

Purification,Crystallization and Some Properties of Lipase from Geotrichum candidum Link

Lipase (EC 3.1.1.3) of Geotrichum candidum Link was purified by means of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, gel-filtration on Sephadex G–100 and Sephadex G–200, and was finally crystallized in concentrated aqueous solution. It was confirmed that the crystallized preparation was homogeneous electrophoretically and ultracentrifugally.

It was estimated with the crystalline enzyme that the sedimentation constant (s₂₀, w) was 4.0, the isoelectric point was pH 4.33, and the molecular weight was 53,000~55,000. From the result of amino acid analysis, none of sulfur containing amino acid was detected in the enzyme. It was also recognized that the crystalline preparation contained about 7% of the carbohydrate and very small amount of lipid. It was characterized that the lipase was the most active at pH 5.6~7.0 on olive oil, at 40°C and was stable in the range of pH 4.2 to 9.8 at 30°C for 24 hr, and was stable below 55°C for 15 min.     

Kallikrein inhibitors in rat plasma.

The kallikrein inhibitor contents of human and animal plasma were determined with glandular kallikreins [EC 3.4.21.8]. One ml of plasma could inactivate 20-700 kallikrein units (KU). Rat plasma was the most potent and inactivated 230-700 KU. However, no enzyme capable of inactivating kallikrein could be found in this plasma. Two fractions which inhibited hog pancreatic kallikrein, a fraction corresponding to alpha2-macroglobulin and a fraction which was eluted prior to albumin, were separated from rat plasma by Sephadex G-200 gel filtration. The former inhibitor could inhibit hog pancreatic kallikrein action on Nalpha-benzoyl-L-arginine ethyl ester (BAEE) as well as in the dog vasodilator assay. The other inhibitor was partially purified from rat plasma. One mg of the preparation inhibited 67 KU and the hydrolysis of 5.8 micronmoles/min of BAEE by hog pancreatic kallikrein [EC 3.4.21.8]. The inhibitor also inhibited other glandular and plasma kallikreins, trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1], etc. The optimal pH of the inhibitor was 7.5-8. The inhibitor was unstable below pH 5, and was destroyed by heating at temperature above 60 degrees. The isoelectric point of the inhibitor was determined by Ampholine focusing to be 4.4, and its molecular weight was estimated to be 73,000 by Sephadex G-100 and G-150 filtrations. Several experimental results suggested that this inhibitor differed from alpha1-antitrypsin.     

Purification and Properties of a Lytic Enzyme from Streptomyces griseus H-402

A lytic enzyme which was capable of lysing cells of Streptococcus mutans was purified from the culture filtrate of Streplomyces griseus H–402 by Amberlite CG–50 treatment, CM-cellulose and hydroxylapatite column chromatographies, and Sephadex G–150 gelfiltration. The lytic enzyme was obtained in a crystalline form which was homogeneous in polyacrylamide gel electrophoresis. The molecular weight was estimated to be 2×10⁴ by the thin-layer gel-filtration method on Sephadex G–75, and 2.3 × 10⁴ by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was found to be a N-acetylmuramidase whose activity was lost by N-bromosuccinimide as an inhibitor.     

Utilization of Synthetic Compounds with Juvenile Hormone Activity for the Silkworm Rearing

A proteinous inhibitor of lipase was purified from soybean cotyledons by the procedures of ammonium sulfate fractionation, gel-filtration on Sephadex G–150 column, DEAE-cellulose column chromatography and isoelectric focusing. The inhibitor obtained by gelfiltration was separated into three active components, D–1, D–2 and D–3, by DEAE-cellulose column chromatography. The D–3 fraction after isoelectric focusing was homogeneous judged from disc electrophoresis. The inhibitory activity was more stable against treatments of heating and Pronase when the D–3 fraction was preincubated with substrate than without substrate. The extent of inhibition was varied by changing the order of addition of reactants and condition of substrate. From these results, the mode of inhibition is discussed.     

Isolation and Crystallization of Microbial Alkaline Protease Inhibitor,S-SI

S–SI, an microbial alkaline protease inhibitor, was produced in the culture broth of Streptomyces albogriseolus S–3253. S–SI was isolated from culture filtrate by salting out with ammonium sulfate, column chromatographies on DEAE-cellulose and Sephadex G–100. S–SI was also isolated by pH adjustment of the effluent from DEAE-cellulose column.

Crystallization of S–SI was carried out, and two distinct shaped crystals, needle shaped and rhombic crystal, were obtained. Homogeneity of these crystals were identified by polyacrylamide disc electrophoresis at various pHs.

S–SI retained its perfect inhibitory activity after treatment at 37°C for 25 hr in the pH range from 3 to 10, and 100°C for 10 min in the pH range from 4 to 6.

From the examination of molecular weight determination with Sephadex G–100, the molecular weight of S–SI was decided to be about 27,000.

S–SI was found to be highly specific towards microbial alkaline proteases.     

Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.     

Detection of Molecular Ions of Cerebroside by Gas Chromatography—Mass Spectrometry

Oxalate oxidase (EC 1.2.3.4) was purified to apparent homogeneity from Pseudomonas sp. OX-53. The molecular weight of the enzyme was about 320,000 by Sephadex G-200 column chromatography and 38,000 by sodium dodecyl sulfate disc electrophoresis. The isoelectric point of the enzyme was pH 4.7 by isoelectric focusing. This enzyme contained 1.12 atoms of manganese and 0.36 atoms of zinc per subunit. Besides oxalic acid, the enzyme oxidized glyoxylic acid and malic acid at lower reaction rates. The Michaelis constant of the enzyme was 9.5 mM for oxalic acid at the optimal pH 4.8. The enzyme was stable from pH 5.5 to 7.0. The enzyme was activated by flavins, phenylhydrazine, and o-phenylenediamine, and inhibited by I^–, Br^–, semicarbazide, and hydroxylamine.     

l-Sorbose Oxidase from Trametes sanguinea

A crude inhibitor for pancreatic lipase was extracted from soybean seeds. The lipase activity decreased curvilinearly with an increase in inhibitor concentration. At a low inhibitor concentration, enhanced inhibition was observed by the co-existence of protein such as bovine serum albumin in the reaction mixture. The lipase activity was inhibited immediately after the addition of inhibitor which did not cause the significant destraction of substrate emulsion. The lipase activities of Aspergillus niger, Rhizopus delemar and castor bean seeds were also inhibited. The inhibition was observed when various oil substrates such as soybean oil, linseed oil, olive oil emulsions and Ediol were used, and the extent of inhibition varied among them. Column chromatography of inhibitor on Sephadex G–100 showed that the molecular weight of a main peak of inhibitor was estimated as about 80,000.     

Penicillin Acylase of Pseudomonas melanogenum KY 3987

Partially purified penicillin acylases (EC 3.5.1.11) were prepared from Pseudomonas melanogenum KY 3987 and Kluyvera citrophila KY 3641 capable of synthesizing d(–)-α-amino-benzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester. As the cell-free extract of P. melanogenum contained high levels of penicillinase (EC 3.5.2.6), the acylase was separated completely from the penicillinase by use of Sephadex column chromatography or electrofocusing. The most salient property of the P. melanogenum penicillin acylase was its substrate specificity to penicillin substrates: it could form 6-APA only from APc but not from penicillin G, penicillin V and p-aminobenzylpenicillin, whereas the K. citrophila acylase acted on all of these penicillins. The P. melanogenum enzyme is hence considered a novel type of penicillin acylase.     

Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β‐galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane‐polyvinyl alcohol (POS‐PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL^?1 initial lactose concentration and 6 U mL^?1 enzyme concentration, obtaining 25.46 ± 0.01 gL^?1 yield of trisaccharides. Although below the HPLC‐IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL^?1 of trisaccharides and 13.79 ± 0.21 gL^?1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady‐state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568–1578, 2017     

l-Asparaginase from E. coli

l-Asparaginase [EC 3.5.1.1], antitumor enzyme, was purified to a crystalline form from the cell free extract of Escherichia coli A-l-3 KY3598, by ethanol fractionation and chromatographies on DEAE cellulose and CM Sephadex. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation: s_{20, w} was 7.87S.

The molecular weight was estimated to be 141,000 by the short column method. The pI of the enzyme protein was 4.75 according to isoelectric electrofocusing.

Amino acid analysis revealed the absence of cysteine or cystine residues in the molecule.

The enzyme exhibited optimal activity between pH 6 and 8. It was stable in the pH range 5.5 ~ 9.0.

The enzyme activity was cleared very slowly in the plasma of dog. Intravenous administration of the enzyme caused a complete regression of the Gardner lymphoma implanted in the C3H mice.     

Synthesis of the mannosyl-O-serine (threonine) linkage of glycoproteins from polyisoprenylphosphate mannose in yeast (Hansenula holstii)

The mannolipid synthesized from GDP-mannose and lipid acceptors in a particulate enzyme preparation from the yeast Hansenula holstii (R. K. Bretthauer, S. Wu, and W. E. Irwin, (1973) Biochim. Biophys. Acta, 304, 736–747) has the properties of dolicholmonophosphate mannose. Transfer of [¹⁴C]mannose from exogenously supplied, purified mannolipid to endogenous protein acceptors of the particulate enzyme fraction has now been demonstrated. The synthesis of radioactive products which are insoluble in chloroform-methanol and water is dependent upon time and concentrations of substrate, particulate fraction protein, and detergent. Addition of MgCl₂ or MnCl₂ to incubation mixtures prepared in the absence of these ions had only small stimulatory effects (20–25%), suggesting that the reaction is not dependent upon metal ions. Relatively high concentrations (0.005 m-0.05 m) of EDTA did partially inhibit the reaction, but this is considered to be due to secondary effects.Seventy percent of the radioactivity in the chloroform-methanol insoluble residue was solubilized with hot, neutral citrate buffer. The Chromatographic properties of this material on Sephadex gels and on DEAE-Sephadex were very similar to the properties of glycoprotein products derived from GDP-[¹⁴C]mannose. The chloroform-methanol insoluble products were also solubilized with Pronase which subsequently resulted in the isolation of a radioactive glycopeptide that contained 25% of the radioactivity transferred from mannolipid. The radioactive component of this glycopeptide was shown by β-elmination experiments and by amino acid analyses to be [¹⁴C]mannose residues linked O-glycosidically to serine and threonine residues. It was concluded, therefore, that one function of the mannolipid is to serve as mannosyl donor in the synthesis of the mannosyl-O-serine (threonine) linkage region of glycoproteins which may be part of the cell wall mannan-protein complex. Other mannose-containing products may also be synthesized from the mannolipid, as β-elimination of the chloroform-methanol insoluble fraction or of the Pronase soluble fraction did not result in recovery of all of the radioactivity as [¹⁴C]mannose.     

Partial Purification of Intra- and Extra-cellular Cellulases from Pyricularia oryzae: with Reference to Optimum pH at Alkaline Side

In order to elucidate the relation between the difference in cellulase activity among various strains of P. oryzae and the optimum pH at alkaline side, and also to know the relation between the intra- and extra-cellulases, the elution patterns of the enzymes from the Sephadex G–100 column were compared and the occurrence of the enzyme fractions showing the optimum pH at alkaline side was investigated.

The elution patterns of the intracellular cellulases were shown to be relatively constant, but those of the extracellular enzymes did not. The peak e appeared comparatively constant, but the peak c was considered to undergo some change during the excretion into the medium.

The optimum pH at alkaline side was shown to occur in the peak e among five peaks on Sephadex G–100 of the partially purified intra- and extra-cellular cellulases. The peak seems to be significant for P. oryzae.     

Purification and Properties of a Chromobacterium Lipase with a High Molecular Weight

A lipase with a high molecular weight was purified from Chromobacterium viscosum by chromatography using the Amberlite CG–50 and Sephadex G–75. The purified lipase (Lipase A) was found to be homogeneous by disc electrophoresis.

Lipase A had an optimum pH around 7 for lipolysis of olive oil and the enzyme was stable at the range of pH 4 to 9 and below 50°C. Zn²⁺, Cu²⁺, Fe³⁺ and high concentrations of l-cysteine, iodoacetic acid and NBS had remarkable inhibitory effects. Bile salts were activator. Lipase A was more active on water insoluble esters than water soluble esters. The isoelectric point of the enzyme was pH 4.7.     

Some Physicochemical Properties and Substrate Specificity of Acid Protease Isolated from Cladosporium sp. No. 45–2

Some physicochemical properties and substrate specificity of crystalline acid protease obtained from Cladosporium sp. No. 45–2 were investigated.

The molecular weight determined by the sedimentation equilibrium method and Sephadex G–75 gel filtration was 32,000 and 30,000, respectively. The isoelectric point was determined as 4.6 by using the Tiselius electrophoresis apparatus and the amino terminal amino acid was found to be alanine. The enzyme did not contain histidine and was composed of 294 amino acid residues. Substrate specificity against synthetic peptides was similar to that of pepsin. The enzyme was remarkably inactivated by a synthetic pepsin inhibitor such as α-Diazo-p-bromo acetophenone, Diazo acetyl glycine ethylester and N-Diazo acetyl norleucine methylester.     

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.