Possible uses of micellar electrokinetic capillary chromatography and high-performance liquid chromatography for the chiral discrimination of some pyrethroids

Micellar electrokinetic capillary chromatography (MECC) and high-performance liquid chromatography (HPLC) were used for the separation of stereoisomers of the lipophilic uncharged pyrethroids cypermethrin, alphamethrin, permethrin, and fenpropathrin. Different kinds of cyclodextrin (β-cyclodextrin, hydroxypropyl-β-cyclodextrin, dimethyl-β-cyclodextrin, and γ-cyclodextrin), surfactants (sodium dodecyl sulphate [SDS] and cetyltrimethylammonium bromide [CTAB]), and cations of background electrolyte (sodium, ammonium, TRIS, and Ammediol) were tested. Optimized conditions (background electrolyte: 50 mmol/l sodium phosphate, pH ≈ 2.5, 150 mmol/l SDS, 150 mg/ml γ-cyclodextrin) allowed the separation of alphamethrin, the eight cypermethrin stereoisomers being eluted in seven peaks and the separation of two enantiomers of fenpropathrin with resolution R_s = 10 and with n ≃ 500,000 theoretical plates. Different experimental conditions, e.g., mobile phase composition, temperature, injected amount, and flow rate, were also optimized in HPLC experiments. The optimal conditions (stationary phase: ChiraDex, 5 μm; mobile phase: 150 mmol triethylamine/l with H₂SO₄ in water (pH = 3.5) with methanol or acetonitrile; flow rate: 0.8 or 0.6 ml/min; temperature: ambient or 30°, 20°, or 10°C; experimental conditions were modified according to the type of analysis) allow chiral discrimination of alphamethrin enantiomers and analysis of permethrin stereoisomers. MECC offers higher efficiency and shorter analysis time than HPLC, but under tested conditions it was shown that the methods complement each other. Chirality 9:162–166, 1997. © 1997 Wiley-Liss, Inc.     

Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine

A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.     

Proteomic analysis of colorectal cancer: discovering novel biomarkers

Colorectal cancer is one of the most common cancers in the Western world. When detected at an early stage, the majority of cancers can be cured with current treatment modalities. However, most cancers present at an intermediate stage. The discovery of sensitive and specific biomarkers has the potential to improve preclinical diagnosis of primary and recurrent colorectal cancer, and holds the promise of prognostic and therapeutic application. Current biomarkers such as carcinoembryonic antigen lack sensitivity and specificity for general population screening. This review aims to highlight the role of current proteomic technologies in the discovery and validation of potential biomarkers with a view to translation to the clinic.     

The Enantiomeric Separation of Tetrahydrobenzimidazoles Cyclodextrins‐ and Cyclofructans

High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) were used to examine the enantiomeric separation of a series of 17 racemic tetrahydrobenzimidazole analytes. These compounds were prepared as part of a synthetic program directed towards a select group of pyrrole‐imidazole alkaloids. This group of natural products has a unique framework of pyrrole‐ and guanidine‐containing fused rings which can be constructed through the intermediacy of a tetrahydrobenzimidazole scaffold. Several bonded cyclodextrin‐ (both native and derivatized) and derivatized cyclofructan‐based chiral stationary phases were evaluated for their ability to separate these racemates via HPLC. Similarly, several cyclodextrin derivatives and derivatized cyclofructan were evaluated for their ability to separate this set of chiral compounds via CE. Enantiomeric selectivity was observed for the entire set of racemic compounds using HPLC with resolution values up to 3.0. Among the 12 different CSPs, enantiomeric recognition was most frequently observed with the Cyclobond RN and LARIHC CF6‐P, while the Cyclobond DMP yielded the greatest number of baseline separations. Fifteen of the analytes showed enantiomeric recognition in CE with resolution values as high as 5.0 and hydroxypropyl‐γ‐cyclodextrin was the most effective chiral additive. Chirality 25:133–140, 2013. © 2012 Wiley Periodicals, Inc.     

高效液相色谱法测定毛豆中吡虫啉残留量

采用高效液相色谱法测定毛豆中吡虫啉农药残留。试样用二氯甲烷超声波提取,中性氧化铝柱净化,石油醚去除脂类杂质,以0.2%冰乙酸水溶液-乙腈(70:30,体积比)为流动相,配备Agilent TC-c18柱、高效液相色谱紫外检测器检测(HPLC-UV),外标法定量。实验表明,毛豆样品中吡虫啉添加回收率为84.0%~102.8%,相对标准偏差(n=6)为6.8%~10.9%,样品中吡虫啉最低检测浓度为0.003 mg/kg。     

Rapid separation of seed gliadins by reversed-phase ultra performance liquid chromatography (RP-UPLC) and its application in wheat cultivar and germplasm identification

To separate gliadin from wheat flour, a novel and stability-indicating reversed-phase ultra performance liquid chromatography (RP-UPLC) method is established and optimized. A comparative analysis of routine capillary electrophoresis (CE), reversed-phase high-performance liquid chromatography (RP-HPLC), and RP-UPLC was performed and the results showed that the resolution and efficiency of RP-UPLC were significantly higher than those of CE and RP-HPLC. Characteristic RP-UPLC patterns of different bread wheat variety and related species were readily identified. These results demonstrated that our RP-UPLC procedure resulted in significant improvements in sensitivity, speed, and resolution, and thus is highly useful in wheat cultivar and germplasm identification.     

Structure and conformational analysis of lipid-associating peptides of apolipoprotein B-100 produced by trypsinolysis

Apolipoprotein B-100 (apo B-100) contains putative lipid-associating regions that are, in part, responsible for its overall structure in human plasma low-density lipoproteins. Some of these regions have been identified by reassembly of the total tryptic peptides of apo B-100 with bovine brain sphingomyelin, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and dimyristoylphos-phatidylcholine (DPMC). Although more than 500 tryptic peptides are predicted from the known number of arginines and lysines in apo B-100, significant amounts of only 13 peptides spontaneously associate with all three phospholipids. These peptides share some structural characteristics, as predicted by several algorithms, that distinguish them from the water-soluble apolipoproteins. Most apolipoproteins associate with lipids via amphipathic helices and are highly helical in native and reassembled lipoproteins. Analysis of all apo B-100 lipophilic peptides by circular dichroism and by use of a predictive algorithm reveals no evidence of amphipathic helices. Although the predictive algorithm suggested that the lipophilic peptides of apo B-100 contain the sequence determinants for -sheet, no spectroscopic evidence for this structure was found. We conclude that the lipophilic regions of apo B-100 liberated by trypsinolysis are highly hydrophobic, although their secondary structures do not fit any simple model.     

Enantioselective and nonenantioselective degradation of organic pollutants in the marine ecosystem

Enantiomeric ratios of 11 chiral environmental pollutants determined in different compartments of the marine ecosystem by chiral capillary gas chromatography and chiral high-performance liquid chromatography allow discrimination between the following processes: enantioselective decomposition of both enantiomers with different velocities by marine microorganisms (α-HCH, β-PCCH, γ-PCCH); enantioselective decomposition of one enantiomer only by marine microorganisms (DCPP); enantioselective decomposition by enzymatic processes in marine biota (α-HCH, β-PCCH, trans-chlordane, cis-chlordane, octachlordane MC4, octachlordane MC5, octachlordane MC7, oxychlordane, heptachlor epoxide); enantioselective active transport through the “blood–brain barrier” (α-HCH); nonenantioselective photochemical degradation (α-HCH, β-PCCH). © 1993 Wiley-Liss, Inc.     

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.     

Analysis of ellagic acid in pomegranate rinds by capillary electrophoresis and high-performance liquid chromatography

Two novel methods for the analysis of ellagic acid in pomegranate (Punica granatum) rinds are proposed. Capillary electrophoresis (CE) was performed in a bare fused-silica capillary using a buffer solution of tri(hydroxymethyl)aminomethane:potassium dihydrogen phosphate (pH 8.4) with an applied voltage of 20 kV and UV detection at 254 nm. HPLC analysis was performed with a Zobax SB C(18) column and a mobile phase consisting of methanol:ethyl acetate:potassium dihydrogen phosphate: phosphoric acid at a flow rate of 1.0 mL/min. Under optimised conditions, the HPLC retention and the CE migration times for ellagic acid were 10.32 and 12.23 min, respectively. Calibration curves of peak area vs. concentration gave correlation coefficients of 0.9999 for HPLC and 0.9990 for CE. The detection limits for HPLC and CE were 2.8 and 2.2 microg/mL, respectively. Average recoveries were 98.32 +/- 1.2% for HPLC and 96.52 +/- 2.8% for CE. Both methods were shown to be suitable for the determination of ellagic acid in pomegranate rinds extraction; however, the CE method required less solvent and gave better column efficiency, whilst the HPLC provided superior precision.     

Fluorescent analysis of alpha-keto acids in serum and urine by high-performance liquid chromatography

A selective procedure using synthetic substrates for determination of exo-1,4,-beta-glucanases in a mixture of exoglucanases , endoglucanases , and beta-glucosidases is formulated. The heterobiosides , p- nithrophenyl -beta-D- cellobioside ( pNPC ) or p-nitrophenyl-beta-D-lactoside ( pNPL ), were used as selective substrates for the measurement of exoglucanase activity. The exoglucanases (especially cellobiohydrolases , which split off cellobiose units from the nonreducing end of the cellulose chain) specifically act on the agluconic bond (between p-nitrophenyl and the disaccharide moiety) and not on the holosidic bond (between the two glucose units of cellobiose). The interfering effect of beta-glucosidase, which acts on both agluconic and holosidic bonds, is overcome by the addition of D-glucono-1,5-delta-lactone, a specific inhibitor of beta-glucosidases. The interference of endoglucanases , which also act on both agluconic and holosidic bonds, can be compensated for by prior standardization of the assay procedure with a purified endoglucanase from the studied mixture of cellulases.     

The transformation of chlorophenols by lactoperoxidase

The lactoperoxidase-catalyzed transformations of penta-, 2,3,4,6,-tetra-, 2,4,6,-tri, 2,4,-di- and 4-monochlorophenol were followed spectrophotometrically. Apparent stoichiometries of chlorophenol: H₂O₂ ranged from 1:1 for the tri- and tetrachlorophenol at pH 7 to 5:2 for pentachlorophenol at pH 4. The initial velocity (ν₀) was only slightly influenced by changes in [H₂O₂] ? 5 μM. ν₀ responded to [chlorophenol] according to the empirical expression ν₀ = [lactoperoxidase]·(k₁[chlorphenol] + k₂[chlorophenol]²). The constant k₁ was found to be 5.8 · 10⁵, 1.8 · 10⁶, 1.9 · 10⁶ M^?1 · s^?1 for the protonated forms of penta-, tetra- and trichlorophenol, respectively, at pH 7. With the di- and monochlorophenol the solution soon became opaque, and the reaction ceased. The results show that more than one reaction occurs. Some comparisons were also made with horseradish peroxidase A and C. Cetyltrimethylammonium bromide prevented opaqueness, but was shown to be a substrate for lactoperoxidase. Assuming an average concentration of 0.1 μM for H₂O₂ and pentachlorophenol in man, the metabolic rate becomes 30 ng/h per g peroxidase-containing tissue, possibly with deposition of the products.     

The 1975 Japanese diet has a stress reduction effect in mice: search for physiological effects using metabolome analysis

Abstract

We aimed to find new physiological effects of the Japanese diet. First, to determine the key components in serum from mice fed the 1975 diet, serum from mice fed the 1960, 1975, 1990 or 2005 Japanese diet was analyzed using CE-TOFMS and LC-TOFMS. Based on these results, the key components were determined by principal component analysis. Among the identified compounds, GABA was included. Therefore, a stress reduction effect was inferred as a novel physiological effect of this diet. Next, we tested whether the 1975 diet had an actual stress reduction effect in mice. Mice were given the 1975 diet or a control diet for 4 weeks, after which they were divided into restraint stress and non-stress groups. Mice fed the 1975 diet had significantly decreased stress parameters compared with those fed the control diet. These results provide the first evidence that the 1975 Japanese diet has a stress reduction effect.     

Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry

Neutral lipids are an important class of hydrophobic compounds found in all cells that play critical roles from energy storage to signal transduction. Several distinct structural families make up this class, and within each family there are numbers of individual molecular species. A solvent extraction protocol has been developed to efficiently isolate neutral lipids without complete extraction of more polar phospholipids. Normal-phase HPLC was used for the separation of cholesteryl esters (CEs), monoalkylether diacylglycerols, triacylglycerols, and diacylglycerols in a single HPLC run from this extract. Furthermore, minor lipids such as ubiquinone-9 could be detected in RAW 264.7 cells. Molecular species that make up each neutral lipid class can be analyzed both qualitatively and quantitatively by on-line LC-MS and LC-MS/MS strategies. The quantitation of >20 CE molecular species revealed that challenging RAW 264.7 cells with a Toll-like receptor 4 agonist caused a >20-fold increase in the content of CEs within cells, particularly those CE molecular species that contained saturated (14:0, 16:0, and 18:1) fatty acyl groups. Longer chain CE molecular species did not change in response to the activation of these cells.     

ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis

Analysis of the long-range architecture of RNA is a challenging experimental and computational problem. Local nucleotide flexibility, which directly reports underlying base pairing and tertiary interactions in an RNA, can be comprehensively assessed at single nucleotide resolution using high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE). hSHAPE resolves structure-sensitive chemical modification information by high-resolution capillary electrophoresis and typically yields quantitative nucleotide flexibility information for 300-650 nucleotides (nt) per experiment. The electropherograms generated in hSHAPE experiments provide a wealth of structural information; however, significant algorithmic analysis steps are required to generate quantitative and interpretable data. We have developed a set of software tools called ShapeFinder to make possible rapid analysis of raw sequencer data from hSHAPE, and most other classes of nucleic acid reactivity experiments. The algorithms in ShapeFinder (1) convert measured fluorescence intensity to quantitative cDNA fragment amounts, (2) correct for signal decay over read lengths extending to 600 nts or more, (3) align reactivity data to the known RNA sequence, and (4) quantify per nucleotide reactivities using whole-channel Gaussian integration. The algorithms and user interface tools implemented in ShapeFinder create new opportunities for tackling ambitious problems involving high-throughput analysis of structure-function relationships in large RNAs.     

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.     

Chiral derivatization for separation of racemic amino and thiol drugs by liquid chromatography and capillary electrophoresis

Separation of racemic amino drugs (α-methylbenzeneethanamine, 6-amino-2-methyl-2-heptanol and 1-aminoethyl-benzenemethanol) and thiol drugs [N-(2-mercapto-1-oxopropyl) glycine, 2-mercaptopropanoic acid, and N-acetyl-3-mercaptovaline] has been evaluated after derivatization. ortho-Phthalaldehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA) were used with either homochiral thiols (N-acetyl-L-cysteine and N-acetyl-D-penicillamine) or amines [(-)-(1R,2S)-norephedrine, L-phenylalanine, L-tyrosine, and 3-hydroxy-L-tyrosine] as chiral selectors according to the analyte reactive group. The resulting 36 diastereoisomeric derivatives were studied using reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Of the CE modes, micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) as surfactant, β-cyclodextrin (β-CD)-modified capillary zone electrophoresis (β-CD-CZE), and β-CD-MEKC were applied. Results highlight respective performance of the reagents and separative techniques. All OPA derivatives of racemic amino drugs were resolved either by MEKC or β-CD-MEKC. In the case of racemic thiol drugs, 10 of the 12 OPA derivatives were resolved in β-CD-CZE. © 1995 Wiley-Liss, Inc.     

Characterization and analysis of the subclasses and molecular species of choline phosphoglycerides from porcine heart by successive chemical hydrolyses and reverse phase high-performance liquid chromatography

Choline phosphoglycerides (CPG) represent the major fraction of heart phospholipids. Since depletion of membrane phospholipids and accumulation of lyso-compounds, particularly lysophosphatidylcholines, have been implicated in arrhythmogenesis, it was of great interest to study the composition of this major phospholipid fraction of the heart at a molecular level in an established animal model. The data presented here describe the first report on the detailed chemical examination of CPG and resolution, characterization and quantitative analysis of the molecular species of this phospholipid fraction from porcine heart by high performance liquid chromatography (HPLC). This fraction constitutes 37.5 ± 0.7% (n = 21) of the total phospholipids and upon successive mild acid and alkaline hydrolyses revealed the presence of essentially three subclasses: diacyl-, alkenylacyl-, and alkylacyl glycerophosphorylcholines, in a relative abundance of 57.7 ± 2.2% (n = 8), 37.3 ± 1.3% (n = 8) and 4.6 ± 0.2% (n = 8), respectively. The fourth subclass, dialkyl CPG was found only in minute amounts (0.43 ± 0.05%, n = 8) and the presence of dialkenyl and alkenylalkyl analogues could not be detected. Alternatively, by converting the CPG fraction to benzoate derivatives after phospholipase C digestion, it was possible to isolate and quantitate subclass composition by TLC/spectroscopy or both subclass compositions and molecular species analysis by HPLC directly by a UV detector online with the column. By these techniques, subclass composition was found to be very similar to that obtained by the chemical hydrolysis technique. By HPLC, up to 25 species can be identified and quantitated in each subclass, their identity being confirmed by gas-liquid chromatography, after their isolation from the column. The analyses showed that up to 74% of the diacyl moiety consisted of 16:0–18:2 (34%), 16:0–18:1 (27%), and 18:0–18:2 (13%) species, while the major species of the alkenylacyl moiety were 16:0–18:2 (44%) 16:0–18:1 (13%), 16:0–20:4 (12%) and 18:1–18:2 (9%) making up more than 75% of the total mass of this subclass. The major molecular species of the alkylacyl moiety was 16:0–18:2, constituting up to 47% of this fraction, while others constituted about 10% (16:0–18:1), 9% (18:1–18:2), 8% (16:0–20:4) and 6% (18:0–18:2), making up 80% of the total mass.The ether chain composition of alkylacyl CPG whether determined after isolation of this fraction by the chemical hydrolysis technique or by HPLC was indistinguishable. Similarly, the aliphatic moieties of diradylglycerols, and their subclasses, whether analysed directly or reconstituted from the molecular species data, were very similar in composition, confirming the accuracy of the data and the reproducibility of the technique devised. This also suggests that this method is suitable to distinguish minor changes in the molecular species of CPG in the heart during the early phase of ischemia and in arrhythmias, and should facilitate further studies on the metabolism of the individual species in health and disease.     

Effective degradation of tannic acid by immobilized rumen microbes of a sika deer (Cervus nippon yesoensis) in winter

In winter seasons, wild sika deer (Cervus nippon yesoensis) inhabiting the Shiretoko Peninsula of Hokkaido Island, Japan, mainly graze woody materials (bark and twigs, etc.) as their feed source. Most of the tree species that they feed upon contain a high level of hydrolysable tannins within the inner bark. Tannins generally lead to low protein digestion and nutrient loss to these herbivorous mammals due to tannization of proteins. In winter months, it is speculated that wild sika deer develop a mechanism to degrade the tannins which are contained in their feed sources, but rumen fluid obtained from sika deer in winter months did not exhibit any ability to degrade tannins in liquid culture medium. However, constant degradation of hydrolysable tannin was observed when Ca-alginate gel beads were used for microbial immobilization and culturing. The gel beads that had been impregnated with 0.6×10⁴ fold-diluted rumen fluid of sika deer in winter and pre-incubated for 24 h under anaerobic conditions supplemented with a 1.5 g/L sugar were reacted with 5 g/L tannic acid solution. Under these conditions, the immobilized rumen bacteria grown in the macrogel beads effectively hydrolyzed tannic acid to release gallic acid monomers. Major bacterial colonies emerging in the Ca-alginate gel beads were identified as Streptococcus macedonicus and this bacterium (EC-D140) was regarded as the most likely candidate as the tannin-degrading bacterium.     

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase. II. Kinetic analysis and identification of scissile bondof prekallikrein in the activation

Activation of human plasma prekallikrein by a bacterial metalloendopeptidase, Pseudomonas aeruginosa elastase, was reported (Shibuya et al. (1991) Biochim. Biophys. Acta 1097, 23–27). Details of the activation process were presently studied. The activation accompanied limited proteolysis of a peptide bond inside of a disulfide bridge of prekallikrein molecule. Amino acid sequencing analysis of the newly generated amino-terminal revealed that the cleavage site was Arg₃₇₁-Ile₃₇₂ bond which is the scissile bond in the activation of prekallikrein with trypsin-type proteinases. A pentapeptide substrate, 2-aminobenzoyl-Ser-Thr-Ile-Val-4-nitrobenzylamide, which contained the amino acid sequence identical to that around the scissile bond of prekallikrein was synthesized. Pseudomonal elastase, indeed, hydrolyzed the substrate at Arg-Ile bond with the kinetic parameters of K_m = 118 μM, k_cat = 1.56/s and k_cat/K_m = 1.33 · 10⁴/s M. These results indicated that the Arg₃₇₁-Ile₃₇₂ bond was sensitive not only to trypsin-type serine proteinases, but also a bacterial metalloproteinase. Kinetic analysis of the prekallikrein activation by psuedomonal elastase, however, revealed that the activation rate was show, though the K_m values was good enough to expect an occurence of this activation in vivo (K_m = 248 nM, k = 6.8 · 10^?4/s, and k_cat/K_m = 2.7 · 10³/s M. The activation rate of prekallikrein by pseudomonal elastase in Hageman factor deficient plasma was remarkably improved when the plasma was reconstituted with purified Hageman factor molecule. From the results, a biologuical significance of the proteinase cascade in the plasma kinin generation was also indicated. The present in vitro study might support the hypothesis that the Hageman factor/kallikrein-kinin system plays an important role in bacterial infection including the pseudomonal one.