CM-Toyopearl 650M Research Articles

Characterization of α-amylase from mandarin orange honey

Summaryα-Amylase was purified from mandarin orange honey by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies, and characterized. The molecular weight of the purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, suggesting that the purified enzyme was a monomer. The purified enzyme exhibited optimum activity at pH 5.0 and at 50°C. Inactivation of the enzyme occurred when the pH was lower than 3.0 or higher than 9.0. The enzyme was activated by Co2+, Mn2+, but inhibited by Hg2+, Mg2+, and Fe3+. By TLC analysis, this enzyme was of the α-type that split the interior α-1,4-glycosidic bonds in a random manner. The relative rate of hydrolysis of the polymeric substrate decreased with decreasing percentage of α-1,4-linkages and with increasing percentage of α-1,6-linkages in substrate similar to the results from commercially available honey.

Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0−7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca 2+ , Mn2+, PCMB, and DTNB, but inhibited by Ba2+, Fe3+, Hg2+, Mg2+, and iodoacetic acid. The purified enzyme was of α -type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α -1,4-linkages and with increasing percentage of α -1,6-linkages in substrate similar to the results from commercially available honey.

Collagen from common minke whale (Balaenoptera acutorostrata) unesu

Collagen was prepared from common minke whale unesu and characterised. The yield of collagen was high, about 28.4% on a wet weight basis. By SDS–PAGE and CM-Toyopearl 650M column chromatography, the collagen was classified as type I collagen. The denaturation temperature of the collagen was 31.5°C, about 6–7°C lower than that of porcine collagen. Attenuated total reflectance-FTIR analysis indicated that acid-soluble collagen from common minke whale unesu held its triple helical structure well, but the structures of porcine skin collagen and pepsin-solubilized collagen from common minke whale unesu were changed slightly, due to the loss of N- and C-terminus domains.

Purification and Characterization of α-Amylase from Honeydew Honey

α-Amylase was extracted from honeydew honey and purified by CM-Toyopearl 650M and Toyopearl HW-55F column chromatographies. The molecular weight of the enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively. The optimum pH was 4.0 and optimum temperature 50°C. Inactivation of the enzyme occured when the pH was higher than 8.0 or lower than 3.0. This enzyme activated by Ca2+ and Mn2+, but inhibited by Fe3+, Hg2+, Zn2+, and EDTA. TLC analysis also suggested that the purified enzyme was of the α-type. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α-1,4-linkages and with increasing percentage of α-1,6-linkages in substrate.

Purification and Partial Characterization of Major Viscous Protein from Yam (Dioscorea opposita Thunb.) Tuber Mucilage tororo

Viscous protein was purified from yam (Dioscorea opposita Thunb.) tuber mucilage tororo by Phenyl-Toyopearl 650M, DEAE-Toyopearl 650M, and CM-Toyopearl 650M column chromatographies, and partially characterized. The molecular weight of the purified protein was estimated to be about 200 kDa, by Native-PAGE and Toyopearl HW-55F gel filtration. Moreover, it had identical subunits of molecular weight of about 32 kDa, either without or with 2-mercaptoethanol, respectively. This protein was identified as dioscorin, the major storage proteins in yam species tubers, when comparing the sequence obtained in an automatic Edman sequencer with the database using FASTA WWW System. On the other hand, the purified protein showed carbonic anhydrase activity and the activity was not inhibited by 2,6-pyridinedicarboxylic acid: zinc atoms were absent in the purified protein. The optimum pH in viscosity was about 5.8 and optimum temperature 20–30°C. Heat denaturation of the protein occurred at about 40°C, but the protein retained more than 66 % of the original viscosity at 70°C for 30 min.

Purification and Characterization of α-Glucosidase I from Japanese Honeybee (Apis cerana japonica) and Molecular Cloning of Its cDNA

alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

Comparison of chromatographic ion-exchange resins: IV. Strong and weak cation-exchange resins and heparin resins

A comparative study was performed on heparin resins and strong and weak cation exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and scanning electron microscopy pictures of chromatographic resins. The resins tested include: Heparin Sepharose FF, SP Sepharose FF, CM Sepharose FF, Heparin Toyopearl 650m, SP Toyopearl 650m, CM Toyopearl 650m, Ceramic Heparin HyperD M, Ceramic S HyperD 20, and Ceramic CM HyperD F. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high isoelectric point (p I), though some decrease of retention with increasing pH was observed for CM Ceramic HyperD F and S Ceramic HyperD 20. Binding of anti-FVII Mab with p I < 7.5 was observed on several resins at pH 7.5. Efficiency results show the expected trend of increasing dependence of the plate height with increasing flow rate of Ceramic HyperD resins followed by Toyopearl 650m resins and the highest flow dependence of the Sepharose FF resins corresponding to their pressure resistance. Determination of particle size distribution by two independent methods, coulter counting and SEM, was in good agreement. Binding strength of cation-exchange resins as a function of ionic strength varies depending on the protein. Binding and elution at high salt concentration may be performed with Ceramic HyperD resins, while binding and elution at low salt concentration may be performed with model proteins on heparin resins. Employing proteins with specific affinity for heparin, a much stronger binding is observed, however, some cation exchangers may still be good substitutions for heparin resins. Dynamic capacity at 10% breakthrough compared to static capacity measurements and dynamic capacity displays that approximately 40–80% of the total available capacity is utilized during chromatographic operation depending on flow rate. A general good agreement was obtained between results of this study and data obtained by others. Results of this study may be used in the selection of resins for testing during protein purification process development.

A new type of aminoacyltransferase from Saccharothrix sp. AS-2 favorable for the synthesis of D-amino acid-containing peptides.

A unique enzyme with some properties favorable for the synthesis of D-amino acid-containing peptides has been purified from the culture broth of Saccharothrix sp. AS-2. The purification steps included ammonium sulfate fractionation, chromatographies on CM-Toyopearl 650M and ProtEx Butyl, and sucrose density-gradient isoelectric focusing. The enzyme, consisting of four subunits of 56 kDa, showed its maximum transfer activity at around pH 8.2 and 35 degrees C, and had an isoelectric point of 5.8. The enzyme yielded homooligomers from methyl esters of D-Asp(OMe), D-Met, D-Phe, D-Trp, D-Tyr, and L-Glu(OMe), but showed no hydrolytic activity toward any of the D- or L-amino acid methyl esters tested. The homooligomers were not formed from the corresponding free amino acids. The reaction of Ac-D-Phe-OMe with DL-Ala-NH(2), DL-Leu-NH(2), DL-Phe-NH(2), or DL-Trp-NH(2) was effectively catalyzed by the enzyme, both the DD- and DL-stereoisomers of the expected N-acetyldipeptide being yielded. The resulting dipeptides remained unhydrolyzed even after 48 h incubation. Also, it showed no detectable hydrolytic activity toward casein, diastereomers of diAla, diMet, and diPhe, D-/L-amino acid amides, or D-/L-amino acid p-nitroanilides, indicating that the enzyme had no peptidase activity leading to secondary hydrolysis of the growing peptide. The enzyme activity was strongly depressed by phenylmethanesulfonyl fluoride, but not by penicillin G or ampicillin, suggesting that the protein is a serine enzyme lacking penicillin-binding ability. These observations lead us to the conclusion that the enzyme from Saccharothrix sp. AS-2 characterized in this study is a new type of aminoacyltransferase with an amino acid ester as the acyl donor, and has potential utility as a catalyst for the synthesis of D-amino acid-containing peptides.

Purification and substrate specificity of honeybee, Apis mellifera L., alpha-glucosidase III.

Alpha-glucosidase III, which was different in substrate specificity from honeybee alpha-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of alpha-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in alpha-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl alpha-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of alpha-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl alpha-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai's) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.

Some properties of a macromolecular conjugate of lysozyme prepared by modification with a monomethoxypolyethylene glycol derivative.

Hen egg-white lysozyme was modified with a succinyl ester derivative of monomethoxypolyethylene glycol (mPEG-COONSu), and some properties of the resulting conjugate (mPEG-lysozyme) were studied. The conjugate was prepared by modification of lysozyme with mPEG-COONSu and purified with use of columns of CM-Toyopearl 650M and Sephadex G-75. Analytical data indicated that in the conjugate, 1.05 moles of mPEG with an average molecular weight of 5,000 were covalently attached to the lysozyme molecule. Tryptic peptide analysis of the conjugate showed that Lys 33 in lysozyme is the residue mainly modified with mPEG-COONSu. Covalent attachment of the mPEG-derivative to amino groups greatly increased the thermostability of lysozyme without any conformational change of the protein molecule. mPEG-lysozyme retained full enzyme activity for glycol chitin, but lytic activity for Micrococcus luteus cells in neutral media was 75% of that of native lysozyme and its optimal pH was at pH 5.0. It was also found that the reactivity of lysozyme with anti-lysozyme antibody from BALB/c mice or human lymphocytes was decreased by modification with mPEG-COONSu. From these findings, it was suggested that mPEG-COONSu can be advantageously used for protein tailoring of lysozyme.

Characterization of Korean native goat lactoferrin

We purified lactoferrin from the colostrum of the Korean native goat ( Capra hircus) by ion-exchange chromatography using CM-Toyopearl 650M followed by affinity chromatography on AF-Heparin Toyopearl 650M. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis suggested the molecular mass of Korean native goat lactoferrin is 82 kDa with an iron saturation of 30% as estimated by spectroscopic analysis. Circular dichroism analysis shows goat lactoferrin molecule contains 24.5%, α-helix; 36.0%, β-structure; 13.5%, β-turn and 26.0%, unordered structure. Heparin binding affinity is the same as that of bovine lactoferrin, but lower than that of human lactoferrin. An analysis using synthetic peptides shows that the peptide from residue 22 to 31—WQRRMRKLGA—exerts a positive heparin-binding ability.

Purification and properties of poly(3-hydroxybutyrate) depolymerase from the fungus Paecilomyces lilacinus D218.

Poly(3-hydroxybutyrate) depolymerase was purified to homogeneity from the culture filtrate of Paecilomyces lilacinus D218 by column chromatography on CM-Toyopearl 650M and hydroxylapatite. The molecular weight of the enzyme was estimated to be 48,000 by SDS-PAGE. Maximal activity was observed near pH 7.0 and 45 degrees C. The Km and Vmax values for PHB were 0.13(mg/ml) and 3750 (U/mg protein), respectively. The enzyme hydrolyzed PHB and p-nitrophenyl fatty acids but not polycaprolactone and triglycerides.

Isolation and Characterization of aβ-Primeverosidase Concerned with Alcoholic Aroma Formation in Tea Leaves

A β-primeverosidase concerned with alcoholic aroma formation during tea processing was purified from cv. Yabukita (Camellia sinensis var. sinensis). The acetone powder was subjected to ammonium sulfate precipitation and then to chromatographic purification with CM-Toyopearl 650M to give three active fractions, Glucosidases (Gls)-I, II, and III fractions. The main Gl-II fraction was characterized as a β-primeverosidase, which hydrolyzed a β-primeveroside into an aglycone and a primeverose (6-O-β-D-xylopyranosyl-β-D-glucopyranose) and was mainly concerned with alcoholic tea aroma formation. The Gl-II fraction was further seperated into Gls-IIa (main) and IIb (minor) by ion-exchange Mono S column chromatography. Gl-IIa was determined to be a monomeric protein of 61 kDa with pI 9.4. Gl-IIa was stable at temperature lower than 45°C and at between pH 5 and 7, and showed its highest activity at 50°C and at pH 5.

Purification and characterization of a new lipase from Fusarium sp. YM-30.

The extracellular lipase from Fusarium sp. YM-30 was purified by a procedure involving ultrafiltration, ammonium sulfate precipitation, and DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Butyl-Toyopearl 650M column chromatographies. The purified lipase was homogeneous with 12kDa of molecular mass by SDS-PAGE, and had high specificities for mono- and diacylglycerols, but low toward triacylglycerols. The enzyme had maximum activity at pH 7.0 to 8.0 and 37 degrees C, and hydrolyzed digalactosyl diglyceride too.

Purification and characterization of a coagulant enzyme, okinaxobin II, from Trimeresurs okinavensis (himehabu snake) venom which releases fibrinopeptides A and B

A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavensis (himehabu snake) venom, releases specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for α-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N-glycanase. Okinaxobin II was much more basic (pI=8.1) than okinaxobin I (pI=5.4). The N-terminal sequence was highly similar to those of okinazobin I and some other snake venom coagulant enzymes such as flavoxobin (Trimeresurus flavoviridis), batroxobin (Bothrops atrox and Bothrops moojeni), and catroxobin (Crotalus atrox). Okinaxobin II hydrolyzed tosyl-L-arginine methly ester and benzoyl-L-arginine p-nitroanilide. The esterase activity was strongly inhibited by diisopropylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethly ketone, indicating that the enzyme is a serine protease like α-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to α-thrombin.

Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp.

Thermostable alkaline protease from an alkaliphilic thermophile Bacillus sp. B18' was purified by using DEAE- and CM-Toyopearl 650M column chromatographies. Molecular weights of the enzyme determined by SDS-PAGE and gel filtration were 30 000 and 28 000, respectively. The optimum pH and temperature toward the hydrolysis of casein were pH 12–13 and 85°C, both of which are higher than those of a mesophilic alkaline protease from an alkaliphile, Bacillus sp. B21-2. The enzyme was stable at pH 5.0–12.0 and about 60% of the initial enzymatic activity was retained after a 60 min incubation period at pH 10.0 and 70°C. Thermostability of the enzyme was enhanced by Ca 2+. The enzyme activity was inhibited by DFP, suggesting that the enzyme is a serine protease. The NH 2-terminal amino acid is Gln, which is that of many subtilisin-type proteases. The 20 residues of the NH 2-terminal amino acid sequence have a comparative high homology with those of other alkaline proteases from alkaliphiles (40–50%), especially thermostable alkaline protease from Bacillus sp. No. AH-101 (95%) and Thermoactinomyces sp. HS682 (95%).

Purification, characterization, and conformational analysis of rabbit plasma lipid transfer protein.

A procedure for rapid isolation of lipid transfer protein (LTP) from commercially available rabbit plasma is described. Use of protease inhibitors was important for obtaining intact, stable LTP. After lipoproteins were precipitated from the plasma by dextran sulfate, column chromatographies through Butyl-Toyopearl 650M, CM-Toyopearl 650M, and Butyl-Toyopearl 650M were employed. Overall purification from plasma was (3830 +/- 710)-fold with a yield of 3-5%. The isolated LTP migrated as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) = 74K and had an NH2-terminal amino acid sequence and amino acid composition closely matching those predicted by its cDNA. This band was recognized by immunoblotting with an anti-human LTP monoclonal antibody, TP2. Gel permeation chromatography revealed that LTP behaved as a globular protein of M(r) = 83K. Isoelectric focusing of the isolated LTP demonstrated a ladder of bands with pI's of 5.7-5.9. The specific activity of rabbit LTP was similar to that of human LTP. Monoclonal antibody TP2, that blocked human plasma LTP activity almost completely, only partially inhibited purified rabbit LTP, and rabbit plasma LTP activity to a similar extent. By a centrifugation binding assay, rabbit LTP was shown to predominantly associate with lipid microemulsion in its presence. Circular dichroism spectroscopy indicated a high content of beta structure, and Provencher and Glöckner analysis gave estimated fractional values of 0.30, 0.39, 0.12, and 0.19 for alpha-helix, beta-sheet, beta-turn, and remainder content, respectively. Upon lipid binding, the helical content did not change drastically, although there was some disordering of beta structure.

Purifications and properties of galactanases from alkalophilic Bacillus sp. S-2 and S-39.

Two galactanases (galactanase S-2 and S-39) were purified to homogeneous states from culture filtrates of alkalophilic Bacillus sp. S-2 and S-39, respectively. The galactanase from Bacillus sp. S-2 were purified by ammonium sulfate precipitation, chromatography on DEAE-Cellulofine, and by gel filtration on Cellulofine GC-200 m. The enzyme had a molecular weight (SDS-PAGE) of 40, 000 and isoelectric point of 8.6. It was most active at pH 10 and 50°C, and was stable between pH 7 and 12 (at 20°C for 15 hr), and below 45°C (at pH 10 for 15min). The galactanase from Bacillus sp. S-39 was purified by ammonium sulfate precipitation and chromatographies on DEAE-Toyopearl 650M and CM-Toyopearl 650M. The enzyne had a molecular weight (SDS-PAGE) of 36, 000 and isoelectric point of 7.1. It was most active at pH 4 and 40°C, and was stable between pH 5 and 12 (at 20°C for 15 hr), and below 60°C (at pH 4 for 15 min). Both enzymes hydrolyzed soybean galactan to produce galactooligosaccharides with very little galactose, but did not attack larch wood arabinogalactan. These enzymes were both referred to as endo-β-1, 4-galactanases (EC 3.2.1.89).

Purification and Characterization of a Coagulant Enzyme, Okinaxobin I, from the Venom of Trimeresums okinavensis (Himehabu Snake) Which Releases Fibrinopeptide B1

A coagulant enzyme, named okinaxobin I, has been purified to homogeneity from the venom of Trimeresurus okinavensis (Himehabu) by chromatographies on Sephadex G-100 and CM-Toyopearl 650M columns. The enzyme was a monomer with a molecular weight of 37,000 and its isoelectric point was 5.4. The enzyme acted on fibrinogen to form fibrin clots with a specific activity of 77 NIH units/mg. Fibrinopeptide B was released at a rate much faster than fibrinopeptide A. The enzyme exhibited 2 to 3 times higher activity toward tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide than bovine thrombin. The esterase activity was strongly inhibited by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like thrombin. The N-terminal sequence was highly homologous to those of coagulant enzymes from T. flavoviridis and Bothrops atrox, moojeni venoms which preferentially release fibrinopeptide A. In order to remove most, if not all, of the bonded carbohydrates, the enzyme was treated with anhydrous hydrogen fluoride (HF), thereby reducing the molecular weight to 30,000. The protein contained approximately 260 amino acid residues when computation was based on this value. The HF-treated enzyme retained about 50% of the clotting and esterolytic (TAME) activities and preferentially released fibrinopeptide B from fibrinogen. The carbohydrate moiety is not crucial for enzyme activity but might be necessary for eliciting full activity.