Amino acids and peptides. XXVIII. Synthesis of peptide fragments related to eglin c and studies on the relationship between their structure and effects on human leukocyte elastase, cathepsin G and alpha-chymotrypsin

Various peptide fragments related to eglin c, which consists of 70 amino acid residues, were synthesized by a conventional solution method and their inhibitory effects on leukocyte elastase, cathepsin G and alpha-chymotrypsin were examined. Among them, H-Arg-Glu-Tyr-Phe-OMe (eglin c 22-25) and H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OMe (eglin c 41-49) inhibited cathepsin G and alpha-chymotrypsin but not leukocyte elastase, while H-Thr-Asn-Val-Val-OMe (eglin c 60-63) inhibited leukocyte elastase but not cathepsin G or alpha-chymotrypsin, although eglin c potently inhibited leukocyte elastase, cathepsin G and alpha-chymotrypsin. These results indicated that the interaction sites of eglin c with leukocyte elastase, cathepsin G and alpha-chymotrypsin might be different.     

Amino acids and peptides. XXX. Synthesis of eglin c (41-49) and eglin c (60-63) and examination of their inhibitory activity towards human leukocyte elastase, cathepsin G, porcine pancreatic elastase and alpha-chymotrypsin

H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OH and H-Thr-Asn-Val-Val-OH, which correspond to the sequences 41-49 and 60-63 of eglin c, respectively, were synthesized by a conventional solution approach using the newly developed 6-chloro-2-pyridyl ester method. The inhibitory activities of the above two peptides against human leukocyte elastase, cathepsin G, porcine pancreatic elastase and alpha-chymotrypsin were examined in comparison with those of the corresponding methyl esters.     

Column separation using Bio-Gel P100 for the characterization of the products of human lung elastin degradation by leucocyte elastase and cathepsin G

The solubilization of human lung elastin by leucocyte elastase and cathepsin G is described. Elastolysis kinetic studies clearly show that leucocyte elastase is more efficient in solubilizing elastin fibres than is cathepsin G. Cathepsin G can degrade elastin but at a much slower rate. Characterization of elastase and cathepsin G soluble elastin fragments, obtained after 24 h of digestion (enzyme-substrate ratio, 1:100), was first performed by isoelectric focusing. Whole digests were focused as 6 bands in a pH range 4.2 to 4.7 and were found to have no significant differences in amino acid compositions. Biogel P-100 gel filtration of the elastase digested fragments separated a major excluded fraction (Mr's: 80,000 to 30,000) and a small retained one (Mr's: 6000 to 4000). Conversely, cathepsin G digests were eluted as a minor excluded fraction and a more important retarded one (Mr's: 6000 to 4000). Only the high molecular weight fractions of both enzymes digests contain crosslinked amino acids; this assigns a role for desmosines in the resistance of elastin to these proteases. These results are discussed in comparison with the data obtained by others.     

Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures. Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated. MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.     

The Level of Hydrophobic Substitution and the Molecular Weight of Amphiphilic Poly-L-lysine-Based Polymers Strongly Affects Their Assembly into Polymeric Bilayer Vesicles

With the aim of producing new materials for drug and gene delivery, the variables associated with the preparation of poly-L-lysine-based vesicles were investigated. Amphiphilic poly-L-lysine graft copolymers with varying levels of grafted methoxypolyethylene glycol (mPEG) and palmitic acid were synthesized using two-step grafting reactions of the macromonomer, mPEG-p-nitrophenyl carbonate (mPEG, MW=5,000), and palmitic acid N-hydroxysuccinimide ester onto poly-L-lysine hydrobromide (MW=4,000 and 19,600). Polymers were characterized by gel permeation chromatography/light scattering. (1)H NMR, and an assay for unreacted varepsilon-amino groups. Polymeric unilamellar vesicles were produced by probe sonication of the amphiphilic poly-L-lysine-based polymers in the presence of cholesterol. Vesicles were characterized by electron microscopy and photon correlation spectroscopy. Vesicle formation was favored by a low molecular weight and a low level of palmitoyl substitution. A vesicle formation index has been derived, F ~ H/L DP, where H is the %molar level of unreacted L-lysine units, L is the %molar level of substituted palmitoyl units, and DP is the square root of the degree of polymerization of the polymer. Additionally, the size of these vesicles may be controlled by controlling the initial molecular weight of the parent poly-L-lysine/resulting amphiphilic polymer. Hence, amphiphilic poly-L-lysine-based polymers of molecular weight=89,000 and 25,000 produced polymeric vesicles of z-average mean diameter 570 nm and 252 nm, respectively. Vesicle encapsulation efficiency for the hydrophilic macromolecule, fluorescein isothiocyanate-dextran (MW=4,400), increased with vesicle size. Copyright 2001 Academic Press.     

Black Bean (Phaseolus vulgaris L.) Polyphenolic Extract Exerts Antioxidant and Antiaging Potential

Phenolic compounds present in common beans (Phaseolus vulgaris L.) have been reported to possess antimicrobial, anti-inflammatory and ultraviolet radiation (UVR) protective properties. UVR from sunlight, which consists of UV-B and UV-A radiations, induces reactive oxygen species (ROS) and free radical formation, consequently activating proteinases and enzymes such as elastase and tyrosinase, leading to premature skin aging. The objective of this work was to extract, characterize and evaluate the antioxidant and antiaging potential of polyphenols from a black bean endemic variety. The polyphenolic extract was obtained from black beans by supercritical fluid extraction (SFE) using CO₂ with a mixture of water–ethanol as a cosolvent and conventional leaching with a mixture of water–ethanol as solvent. The polyphenolic extracts were purified and characterized, and antioxidant potential, tyrosinase and elastase inhibitory potentials were measured. The extract obtained using the SFE method using CO₂ and H₂O–Ethanol (50:50 v/v) as a cosolvent showed the highest total phenolic compounds yield, with 66.60 ± 7.41 mg GAE/g coat (p > 0.05) and 7.30 ± 0.64 mg C3GE/g coat (p < 0.05) of anthocyanins compared to conventional leaching. Nineteen tentative phenolic compounds were identified in leaching crude extract using ESI-QTOF. Quercetin-3-D-galactoside was identified in crude and purified extracts. The purified SFC extract showed IC₅₀ 0.05 ± 0.002 and IC₅₀ 0.21 ± 0.008 mg/mL for DPPH and ABTS, respectively. The lowest IC₅₀ value of tyrosinase inhibition was 0.143 ± 0.02 mg/mL and 0.005 ± 0.003 mg/mL of elastase inhibition for leaching purified extract. Phenolic compounds presented theoretical free energy values ranging from −5.3 to −7.8 kcal/mol for tyrosinase and −2.5 to −6.8 kcal/mol for elastase in molecular docking (in silico) studies. The results suggest that the purified extracts obtained by SFE or conventional leaching extraction could act as antioxidant and antiaging ingredients for cosmeceutical applications.     

Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken.

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23,500, 25,700 and 25,000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and k(cat)/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.     

Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA

A solvent tolerant Pseudomonas aeruginosa PseA strain was isolated from soil. It secreted a novel alkaline protease, which was stable and active in the presence of range of organic solvents, thus potentially useful for catalysis in non-aqueous media. The protease was purified 11.6-fold with 60% recovery by combination of ion exchange and hydrophobic interaction chromatography using Q-Sepharose and Phenyl Sepharose 6 Fast Flow matrix, respectively. The apparent molecular mass based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was estimated to be 35,000 Da. The enzyme was stable in the pH range of 6.0-9.0, the optimum being 8.0. The Km and Vmax towards caseinolytic activity were found to be 2.7 mg/ml and 3 micromol/min, respectively. The protease was most active at 60 degrees C and characterized as a metalloprotease because of its sensitivity to EDTA and 1,10-phenanthroline. It was tested positive for elastase activity towards elastin-orcein, thus appears to be an elastase, which is known as pseudolysin in other strains of P. aeruginosa. The protease withstands range of detergents, surfactants and solvents. It is stable and active in all the solvents having log P above 3.2, at least up to 72 h. These two properties make it an ideal choice for applications in detergent formulations and enzymatic peptide synthesis.     

Amino acids and peptides. XXIX. Synthesis of peptide fragments related to active center of eglin c and studies on the relationship between their structure and their inhibitory activity against cathepsin G and alpha-chymotrypsin

H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OMe, corresponding to the sequence 41-49 of eglin c, inhibited human leukocyte cathepsin G and alpha-chymotrypsin. In order to gain further insight into the relationship between the structure and the inhibitory activity against cathepsin G and alpha-chymotrypsin, peptide fragments related to the above nonapeptide were synthesized by a conventional solution method and their inhibitory activities were examined. The smallest peptide which exhibited inhibitory effects on the above enzymes was H-Pro-Val-Thr-Leu-OMe, corresponding to the sequence 42-45 of eglin c.     

Synthesis of a heptacontapeptide corresponding to the entire amino acid sequence of eglin C

A heptacontapeptide corresponding to the entire amino acid sequence of eglin c was synthesized by the conventional solution method using a minimal protecting method. The synthetic eglin c exhibited a symmetrical single peak on HPLC at the same retention time as an authentic eglin c, and had the same inhibitory activity against human leukocyte elastase, cathepsin G and alpha-chymotrypsin (Ki = 6.0 x 10(-9) M, 5.5 x 10(-9) M and 2.5 x 10(-9) M, respectively) as N alpha-acetyl-eglin c synthesized genetically (Ki = 5.1 x 10(-9) M, 1.5 x 10(-8) M and 2.2 x 10(-9) M, respectively).     

Purification, subunit structure and inhibitor profile of cathepsin A.

Cathepsin A (EC 3.4.16.1), a lysosomal carboxypeptidase, has been purified 1374-fold from pig kidney. Purification steps included concanavalin A-Sepharose and phenyl-Sepharose chromatography and chromatofocusing. The specific activity (16.9 U/mg) of the purified enzyme was significantly higher than previously reported values. The enzyme preparation appeared homogeneous when analyzed by non-denaturing polyacrylamide gel electrophoresis and was free of detectable protease contamination. The molecular mass (M(r) = 97,000), isoelectric point (5.0), and sensitivity to inhibitors were consistent with reported properties of cathepsin A. However, the previously reported three-peptide chain structure was not observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol demonstrated that the enzyme is composed of two M(r) 47,000 subunits, each of which dissociate in the presence of 2-mercaptoethanol into two polypeptide chains of 19,000 and 31,000.     

One‐step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X‐100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio‐active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.     

Effects of pH and phosphate on the adsorptive fractionation of purified Aldrich humic acid on kaolinite and hematite

The molecular weight (MW) fractionation of purified Aldrich humic acid (PAHA) resulting from adsorption on kaolinite and hematite was investigated for different solution pH and phosphate conditions. Adsorption was highly pH-dependent, with higher uptake at lower pH values. For all pH conditions, the weight-average MW (MWw) of residual PAHA remaining in solution after adsorption deviated from the original MWw, indicating that preferential adsorption of certain MW components occurred. The extent of preferential adsorption depended on the percent carbon adsorption at a given pH condition. For similar percent carbon adsorption ranges, a greater extent of preferential adsorption of the higher MW PAHA components was observed with higher pH values as demonstrated by the lowest residual MWw value occurring at pH 9. Detailed analyses of selected residual PAHA samples clearly showed that adsorption selectivity for particular MW components was strongly influenced by solution pH. The extent of preferential adsorption of lower MW PAHA components decreased in the presence of a small amount of phosphate. This effect was more evident for hematite than kaolinite, and became greater with lower solution pH irrespective of the mineral type. The different fractionation patterns observed for PAHA were reasonably well explained by the physicochemical trends occurring in its MWw fractions and the underlying sorption processes.     

Ga-labeling of immunoglobulin G with high specific radioactivity

To obtain radiogallium labeled immunoglobulin G with a high specific radioactivity for in vitro use, a 67Ga source was purified by extraction from 67Ga-gallium citrate with butyl acetate, and a 67Ga-labeling solution was produced. This solution was then used to label a deferoxamine-immunoglobulin G conjugate. Both a very high specific radioactivity (872 +/- 56 MBq/mg) and a high labeling efficiency (94.0%) were achieved.     

Purification and Characterization of Paraoxonase 1 (PON1) from Swiss Black,Holstein, and Montofon Bovines

Paraoxonase 1 (PON1: EC 3.1.8.1) is a calcium-dependent enzyme associated with high-density lipoproteins (HDLs) and has a protective effect against oxidation of low-density lipoproteins (LDLs) in mammals. PON1 is the best-studied member of a family of enzymes called serum paraoxonases, or PONs, identified in mammals and other vertebrates as well as in invertebrates. PONs exhibit a range of important activities, including drug metabolism and detoxification of organophosphates such as nerve agents. This study reports, for the first time, purification and biochemical characterization of serum PON1 from different bovine breeds namely Swiss Black, Holstein, and Montofon. Bovine serum PON1s were purified using ammonium sulfate precipitation followed by Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS–polyacrylamide gel electrophoresis of the purified enzymes indicates a single band with an apparent MW of 43 kDa. The purified enzymes had a specific activity of 10.78, 27.00, and 22.38 U/mg for Swiss Black, Holstein, and Montofon bovines, respectively. The overall purification rates of our method were 262.47-, 2,476.90-, and 538.06-fold for Swiss Black, Holstein, and Montofon bovines, respectively. Furthermore, using phenyl acetate as a substrate, we determined the K _M and V _max values of the purified enzymes, as 0.80 mM, 1428.5 U/ml for Swiss Black; 0.40 mM, 714.3 U/ml for Holstein; and 0.50 mM, 1,111.1 U/ml for Montofon bovine. The present study has revealed that there is no substantial difference in PON1 activities among the studied bovine breeds.     

Purification and Characterization of Extracellular Inulinase from a Marine Yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and Inulin Hydrolysis by the Purified Inulinase

The extracellular inulinase in the supernatant of the cell culture of the marine yeast Cryptococcus aureus G7a was purified to homogeneity with a 7.2-fold increase in specific inulinase activity compared to that in the supernatant by ultrafiltration, concentration, gel filtration chromatography (Sephadex™ G-75), and anion exchange chromatography (DEAE sepharose fast flow anion exchange). The molecular mass of the purified enzyme was estimated to be 60.0 kDa. The optimal pH and temperature of the purified enzyme were 5.0 and 50 °C, respectively. The enzyme was activated by Ca²⁺, K⁺, Na⁺, Fe²⁺, and Zn²⁺. However, Mg²⁺, Hg²⁺, and Ag⁺ acted as inhibitors in decreasing the activity of the purified inulinase. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1,10-phenanthroline. The K _m and V _max values of the purified enzyme for inulin were 20.06 mg/ml and 0.0085 mg/min, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified inulinase, indicating the purified inulinase had a high exoinulinase activity.     

Ligand location and biochemical productivity of silica-based immunoaffinity adsorbents.

Ligand location within particles, detected by immunogold labelling, was shown to influence the biochemical productivity of a silica-based solid phase, Sorbsil C-500, using a model ligand-biomolecule system (immobilised human immunoglobulin G-anti-human immunoglobulin G monoclonal antibody). The distribution of the ligand was in turn affected by the initial ligand challenge used to prepare the immunoadsorbents. Maximal productivity was achieved with adsorbents prepared with an initial challenge of about 3 mg human immunoglobulin G per ml: the ligand in these cases was shown to be more uniformally distributed within the adsorbent particles than adsorbents, exhibiting low productivity, prepared with either low (1 mg/ml) or high (9 mg/ml) concentrations of human immunoglobulin G. The ligand in the latter was restricted to the periphery of the particles.     

A simplified method for the preparation of WGA-Sepharose 4B and its use in the purification of MN blood group antigens

A simplified method for the preparation of wheat germ agglutinin(WGA)-Sepharose 4B by coupling highly purified WGA, prepared by improved affinity chromatography, with BrCN activated Sepharose 4B in a solution of high carbonate buffer is described. The amount of WGA linked to Sepharose 4B was 82.40% (3.07 mg WGA per ml Sepharose 4B). MN blood group antigens of human erythrocyte membranes purified with WGA-Sepharose 4B affinity chromatography showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The yield of the antigens from 400 mL fresh blood was 32-40 mg. The WGA-Sepharose 4B column could be used several times without loss of activity.     

High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver.

A high-performance liquid chromatographic procedure for the isolation of the three cysteine proteinases, namely cathepsins B, H and L, is described. The method is based on the following four steps. (1) A classical AcA 44 gel permeation separation with a 30-70% ammonium sulphate fraction from the human liver homogenate is used to remove the non-enzymic high-molecular-mass components. (2) Preparative cation-exchange chromatography on a CM-SW TSK column can separate the three proteinases. (3) An anion-exchange step on a semi-preparative DEAE-SW TSK column for the cathepsin H fraction is used to remove a small amount of cathepsins B and L activities. (4) The three separated enzymes are purified on an analytical TSK gel 2000 SW column. The purity of each enzyme is assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrofocusing on polyacrylamide gels. To check the activities of the purified proteinases, the kinetic constants [Michaelis constant (KM) and catalytic constant (Kcat)] and the ratio Kcat/KM against the fluorigenic substrates Arg-NH-Mec, Z-Arg-Arg-NH-Mec and Z-Phe-Arg-NH-Mec after active-site titration using E-64, were determined. Z-Phe-Phe-CNH2 was also used as a specific inhibitor of cathepsin L. This method requires only 6 g of human liver, and gives a high yield of the three lysosomal cysteine-proteinases: thus, about 150 micrograms of cathepsin B and 50 micrograms each of cathepsins L and H are obtained in a single run.     

Novel two-dimensional "ring and chain" morphologies in Langmuir-Blodgett monolayers of PS-b-PEO block copolymers: effect of spreading solution concentration on self-assembly at the air-water interface

A polystyrene-b-poly(ethylene oxide) (PS-b-PEO) (MW = 141k, 11.4 wt% PEO) diblock copolymer in the hydrophobic regime was spread from chloroform solutions of various concentrations at the air-water interface, and the resultant monolayers were transferred to glass substrates and imaged using atomic force microscopy. Monolayers prepared under identical conditions were also characterized at the air-water interface via Langmuir compression isotherms. The effects of spreading solution concentration on surface features, compressibility, and limiting mean molecular area were determined, revealing several interesting trends that have not been reported for other systems of PS-b-PEO. Spreading solutions > or = 0.50 mg/mL resulted almost exclusively in dot and spaghetti morphologies, with no observed continent features, which have been commonly found in more hydrophobic systems. For lower spreading solutions, < or = 0.25 mg/mL, we observed a large predominance of two novel surface morphologies, nanoscale rings and chains. The surface pressure (pi)-area (A) isotherms also exhibited a unique dependence on the spreading solution concentration, with limiting mean molecular areas and isothermal compressibilities of PS-b-PEO monolayers increasing below a critical concentration of spreading solution, suggesting a greater contribution from the PEO blocks. These results suggest that PS chain entanglement prior to solvent evaporation plays an important kinetic role in the extent of PEO adsorption at the air-water interface and in the morphologies of the resulting self-assembled surface aggregates.