Gel Filtration HPLC Research Articles

Thiol oxidation and di-tyrosine formation in human plasma proteins induced by inflammatory concentrations of hypochlorous acid

In this study, we assessed the oxidative damage occurring in plasma proteins when human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). We used specific thiol labelling and Western blot analyses to determine protein thiol oxidation, as well as analytical gel filtration HPLC coupled to fluorescence detection to explore formation of high molecular weight (HMW) protein aggregates. Thiol-containing proteins oxidized by HOCl were identified by redox proteomics. Mass spectrometry (MS) analysis was performed to elucidate the protein composition of HMW aggregates. α1-antitrypsin, transthyretin, and haptoglobin showed thiol oxidation at HOCl concentrations higher than those causing complete oxidation of albumin. At the highest HOCl concentrations, formation of carbonylated and di-tyrosine cross-linked HMW protein aggregates also occurred. MS analysis identified fibrinogen, complement C3 and apolipoprotein A-I as components of HMW protein aggregates. These results could be relevant for human diseases characterized by inflammatory conditions in which myeloperoxidase and HOCl are involved. Biological significanceIn this study we evaluated the oxidative damage occurring on plasma proteins when reconstituted human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). Pathophysiological concentrations of HOCl are able to induce different modifications on plasma proteins such as carbonylation, sulfhydryl oxidation and formation of high molecular weight (HMW) protein aggregates characterized by di-tyrosine fluorescence. There are two relevant aspects emerging from this paper. The first one consists on identifying low abundant proteins undergoing sulfhydryl oxidation by biotin-maleimide derivatization followed by MALDI-TOF mass spectrometry. This approach suggests three low-abundant proteins undergoing HOCl-induced oxidation: transthyretin, α1-antitrypsin, and haptoglobin. In addition, we analysed HMW protein aggregates forming after HOCl exposure. These aggregates are characterized by carbonylation, intra- and/or intermolecular di-tyrosine bridges. After their isolation from SDS-PAGE gel electrophoresis, using electrospray tandem mass spectrometry coupled to reversed-phase nanoscale capillary liquid chromatography, we identified some protein constituents of these HMW aggregates such as α, β, γ fibrinogen chains, apolipoprotein A-I and complement C3. In particular, our work highlights how fibrinogen is an important constituent of HOCl-induced HMW protein aggregates validating the mass spectrometry result with additional experiments. Further investigations are required in order to evaluate the possibility to use carbonylated and di-Tyr cross-linked HMW protein aggregates as (early) biomarkers for disease progression in inflammatory conditions in which myeloperoxidase and HOCl are involved.

Metal Nanoparticles Dispersion By Chaperonin Complexes

Introduction GroEL is the best characterized chaperonin; it is found in the cytoplasm of Escherichia coli and is essential for cell viability and growth. GroEL is a large cylindrical protein complex comprising two heptameric rings of identical 57-kDa subunits, and these rings are stacked back to back. GroES is a single heptameric ring of identical 10-kDa subunits. GroEL encapsulates the substrates to the cavities, GroEL binds a wide variety of substrate proteins in non-native states, and forms a binary complex, which then binds seven ATP molecules with positive cooperativity and GroES to the same (cis) GroEL ring, to form the cis-ternary complex. The binding of GroES induces the encapsulation of the substrate protein into an enlarged cavity (horizonal width of 5-6nm and vertical width of 6-7nm) inside the cis-ring, which is capped by GroES. In the cis-cavity, non-native protein initiates folding without the risk of aggregation. Following hydrolysis of ATP in the cisring, the products of this cycle, ADP and folded substrate protein, are released with GroES, as another similar folding-active assembly is formed in the opposite trans ring. On the other hand, the hydrolysis-defective mutant GroEL(D52/398A) can bind two GroES in the presence of ATP, producing a symmetric 1:2 GroEL-GroES complex (the “football”-shaped complex) with half-time of ~150 h (~6 days). The property of the GroEL(D52/398A) looks beneficial for dispersing aggregative nanoparticles and hydrophobic compounds. FePt, Pt, CdSe/ZnS and Au were used to encapsulate in GroEL(D52/398A) as metal nanoparicles because these nanoparticles are used to engineering manufactures such as semiconductors, devices and batteries. Experiments Pt (average φ2 nm), FePt (φ4 nm), CdSe/ZnS (φ7nm and 6 nm) and Au (φ10 nm, 4 nm and 2 nm) were used to encapsulate in GroEL(D52/398A) as metal nanoparicles. In the case of nanoparticle encapsulation in one side of GroEL(D52/398A) cavities, 0.1-1.0 µM GroEL(D52/398A), 0.2-2.0 µM GroES (2-fold mol of GroEL), and 1 mM ATP were added to the sonicated nanoparticle solution. For two different nanoparticle encapsulation in both sides of GroEL(D52/398A) cavities, GroEL(D52/398A) which is bound to first nanoparticle was added to the same molar of GroES and 5 mM ADP. The bullet-shaped GroEL(D52/398A) encapsulating first nanoparticle was mixed with the second nanoparticle solution for 1 minute, then, GroES and 1 mM ATP were added to form the football-shaped complex. GroEL(D52/398A) complexes were stained with 1% phosphotungstic acid (pH 4.0) for the observation with the transmission electron microscopy JEM 2000EX or JEM 2100 (JEOL Co., Ltd.). For protein crystalizaion, the sample solution of metal nanoparticle encapsulating GroEL(D52/398A) complexes was added to 1% ammonium molybdate/0.2 % PEG1500 with equal amount volume. Then, the mixture was dropped on TEM grid or flesh cleaved mica and air dried. Fixed protein was observed using with TEM or scanning probe microscopy SPM 9600 (Shimazu Corp.). Analysis was performed using with gel filtration HPLC system equipped with a G3000SWXL (TOSOH) and elution buffer which was consisted of 20 mM HEPES/KOH (pH 7.0), 100 mM KCl, 5 mM MgCl2 and 50 mM Na2SO4 at a flow rate of 0.5 ml/min. The eluate was monitored using a UV detector set at the wavelength 280 nm. Fluorescence intensity was detected at an excitation wavelength of 370 nm and emission at 490 nm for CdSe/ZnS λ490 or 575 nm emission wavelength for CdSe/ZnS λ575 was used. Results and discussion From TEM images, the highest FePt nanoparticles encapsulation percentage of GroEL(D52/398A) was about 95% in this experimental condition. Pt, CdSe/ZnS and Au nanoparticles were encapsulated in GroEL(D52/398A) complexes, too. GroEL(D52/398A) wasn’t seemed to select metal nanoparticle. Two CdSe/ZnS nanoparticles encapsulation of GroEL(D52/398A) of analysis indicated FL emission peaks of both wave length at 490 nm and 575 nm around 12 min of retention time which GroEL(D52/398A) elution time with UV detector. This result showed a possibly that GroEL(D52/398A) could encapsulate two particles in each their cavities. In a presence of protein crystallization reagent, metal nanoparticles encapsulating GroEL(D52/398A) complexes showed tendency to fill up with themselves on TEM grid or mica as an experimental flat plane. These experiments are continued to make monolayer aligns of metal nanoparticle encapsulating GroEL(D52/398A) complexes on flat plated of manufactures. These results from a sequential experiments of GroEL(D52/398A) suggest a potential that GroEL(D52/398A) will support to construct nano manufactures.

Identification of SER-PRO-CYS Peptide in Blood Serum of Multiple Sclerosis Patients.

Monitoring of multiple sclerosis (MS) requires additional molecular markers. Recently, we used original TCA-precipitation/extraction approach in combination with MALDI TOF/TOF mass-spectrometry and identified earlier unknown 48 kDa form of the unconventional myosin IC isoform b (Myo1C) in blood serum of the MS patients. Further examination of TCA-extracted fraction of blood serum of these patients by means of thin-layer chromatography and HPLC gel-filtration allowed detecting 300-500 Da peptides. MALDI TOF/TOF massspectrometry of these peptides showed that they contain Ser-Pro-Cys amino acid sequence. We discussed potential mechanisms of a release of these peptides that were earlier unknown in blood serum of the MS patients.

Thailandins A and B, New Polyene Macrolactone Compounds Isolated from Actinokineospora bangkokensis Strain 44EHW(T), Possessing Antifungal Activity against Anthracnose Fungi and Pathogenic Yeasts.

Two new polyene macrolactone antibiotics, thailandins A, 1, and B, 2, were isolated from the fermentation broth of rhizosphere soil-associated Actinokineospora bangkokensis strain 44EHW(T). The new compounds from this strain were purified using semipreparative HPLC and Sephadex LH-20 gel filtration while following an antifungal activity guided fractionation. Their structures were elucidated through spectroscopic techniques including UV, HR-ESI-MS, and NMR. These compounds demonstrated broad spectrum antifungal activity against fungi causing anthracnose disease (Colletotrichum gloeosporioides DoA d0762, Colletotrichum gloeosporiodes DoA c1060, and Colletotrichum capsici DoA c1511) as well as pathogenic yeasts (Candida albicans MT 2013/1, Candida parasilopsis DKMU 434, and Cryptococcus neoformans MT 2013/2) with minimum inhibitory concentrations ranging between 16 and 32 μg/mL. This is the first report of polyene antibiotics produced by Actinokineospora species as bioactive compounds against anthracnose fungi and pathogenic yeast strains.

Structure elucidation of two novel yak milk oligosaccharides and their DFT studies

Milk is a primary dynamic biological fluid responsible for development of neonates. Besides the other regular constituents it have oligosaccharides in it which are responsible for antitumor, anticancer, antigenic and immunostimulant activities. In our endeavor to find biologically active novel oligosaccharides, yak milk was taken, which is a rich source of oligosaccharide and its milk is used as antihypertensive, antioxidative and heart strengthening agent in folk medicine. For this purpose yak milk was processed by method of Kobata and Ginsburg followed by gel filtration HPLC and CC which resulted in the isolation of two novel milk oligosaccharides namely (I) Grunniose and (II) Vakose. The structure of purified milk oligosaccharides were elucidated with the help of chemical degradation, chemical transformation, spectroscopic techniques like NMR (1H, 13C and 2D-NMR), structure reporter group theory and mass spectrometry. The optimized geometry of compound Grunniose and Vakose, at B3LYP method and 6-311 + G basis set on Gaussian 09 program, show that the compound Grunniose is lower in energy as compared to compound Vakose.Compound Grunniose -▪Compound Vakose -Glc-β(1→3) Gal-β(1→3) GlcNAc-β(1→6) Gal-β(1→3) GlcNAc-β(1→3) Gal-β(1→4) Glc

Purification of glycocalicin from human plasma.

Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product.

Preparation of proteoglycan from salmon nasal cartilage under nondenaturing conditions.

Salmon nasal cartilage was micronized in ethanol using a rotor-stator homogenizer for the high yield of proteoglycan extraction. This procedure also brought about depressing the degradation of proteoglycan and the contamination of collagens. Proteoglycan was extracted by 4 M magnesium chloride and isolated by anion-exchange chromatography. The gel filtration HPLC and the antibody reactivity showed that the core protein was intact.

Two Isorhamnetin Glycosides from Arthrocnemum glaucum that Inhibit Adipogenesis in 3T3-L1 Adipocytes

In our search for bioactive compounds from North African plants [1, 2], we found that the Tunisian medicinal plant Arthrocnemum glaucum Delile inhibited adipogenesis in 3T3-L1. A. glaucum, which belongs to the Chenopodiaceae, is a densely branched robust plant that grows wild in the Sahara Desert of Tunisia, especially in the Mediterranean Coastal region. It was previously reported that some alkaloids and some sterols were isolated from A. glaucum [3–6]. In this research, the bioassay-guided fractionation of this plant implied that the bioactive components are two isorhamnetin glycosides, isorhamnetin-3-O-[apiosyl(1 6)]glucosyl-7-O-rhamnoside (1) and isorhamnetin-3-O-rutinoside (2). In this paper, we report the isolation and structure elucidation of the two isorhamnetin glycosides 1 and 2 from aerial parts of this plant and their antiadipogenesis activity. The aerial part of A. glaucum was collected at the Sahara Desert of Tunisia. The voucher specimen (TPM-20080401) is maintained at Alliance for Research on North Africa (ARENA), University of Tsukuba and Environmental Bioprocesses Laboratory at Biotechnology Center of Sfax. The leaves were dried in the shade at 25 C. The dried leaves of A. glaucum (100 g) were extracted with MeOH. The MeOH extracts were partitioned between EtOAc and H2O, and the H2O layer was partitioned with n-BuOH. The n-BuOH-soluble portion (1.33 g) was subjected to ODS column chromatography and purified by reversed-phase HPLC or gel-filtration HPLC to afford two known flavonol glycosides, isorhamnetin-3-O[apiosyl(1 6)]glucosyl-7-O-rhamnoside (1, 0.0085%) and isorhamnetin-3-O-rutinoside (2, 0.0027%). Structure elucidation of 1 and 2 was through spectroscopic data, mainly 1H and 13C NMR spectra and ESI-MS spectra. Full assignment of signals in the NMR spectra of 1 and 2 was confirmed by 2D NMR experiments (1H–1H COSY, NOESY, HMQC, and HMBC) and comparison with reported data [7, 8].

A central role for intermolecular dityrosine cross-linking of fibrinogen in high molecular weight advanced oxidation protein product (AOPP) formation

BackgroundAdvanced oxidation protein products (AOPPs) are dityrosine cross-linked and carbonyl-containing protein products formed by the reaction of plasma proteins with chlorinated oxidants, such as hypochlorous acid (HOCl). Most studies consider human serum albumin (HSA) as the main protein responsible for AOPP formation, although the molecular composition of AOPPs has not yet been elucidated. Here, we investigated the relative contribution of HSA and fibrinogen to generation of AOPPs. MethodsAOPP formation was explored by SDS-PAGE, under both reducing and non-reducing conditions, as well as by analytical gel filtration HPLC coupled to fluorescence detection to determine dityrosine and pentosidine formation. ResultsFollowing exposure to different concentrations of HOCl, HSA resulted to be carbonylated but did not form dityrosine cross-linked high molecular weight aggregates. Differently, incubation of fibrinogen or HSA/fibrinogen mixtures with HOCl at concentrations higher than 150μM induced the formation of pentosidine and high molecular weight (HMW)-AOPPs (>200kDa), resulting from intermolecular dityrosine cross-linking. Dityrosine fluorescence increased in parallel with increasing HMW-AOPP formation and increasing fibrinogen concentration in HSA/fibrinogen mixtures exposed to HOCl. This conclusion is corroborated by experiments where dityrosine fluorescence was measured in HOCl-treated human plasma samples containing physiological or supra-physiological fibrinogen concentrations or selectively depleted of fibrinogen, which highlighted that fibrinogen is responsible for the highest fluorescence from dityrosine. ConclusionsA central role for intermolecular dityrosine cross-linking of fibrinogen in HMW-AOPP formation is shown. General significanceThese results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.

18F-glutathione conjugate as a PET tracer for imaging tumors that overexpress L-PGDS enzyme.

Lipocalin-type prostaglandin D synthase (L-PGDS) has been correlated with the progression of neurological disorders. The present study aimed at evaluating the imaging potency of a glutathione conjugate of fluorine-18-labeled fluorobutyl ethacrynic amide ([18F]FBuEA-GS) for brain tumors. Preparation of [18F]FBuEA-GS has been modified from the -4-tosylate derivative via radiofluorination in 5% radiochemical yield. The mixture of nonradioactive FBuEA-GS derived from a parallel preparation has be resolved to two isomers in a ratio of 9∶1 using analytic chiral reversed phase high performance liquid chromatography (RP-HPLC). The two fluorine-18-labeled isomers purified through nonchiral semipreparative RP-HPLC as a mixture were studied by assessing the binding affinity toward L-PGDS through a gel filtration HPLC, by analyzing radiotracer accumulation in C6 glioma cells, and by evaluating the imaging of radiotracer in a C6 glioma rat with positron emission tomography. The inhibition percentage of the production of PGD2 from PGH2 at the presence of 200 µM of FBuEA-GS and 4-Dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)butyl]piperidine (AT-56) were 74.1±4.8% and 97.6±16.0%, respectively. [18F]FBuEA-GS bound L-PGDS (16.3–21.7%) but not the isoform, microsomal prostaglandin E synthase 1. No binding to GST-alpha and GST-pi was observed. The binding strength between [18F]FBuEA-GS and L-PGDS has been evaluated using analytic gel filtration HPLC at the presence of various concentrations of the cold competitor FBuEA-GS. The contrasted images indicated that the radiotracer accumulation in tumor lesions is probably related to the overexpression of L-PGDS.

Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan

Biological activities of salmon nasal cartilage proteoglycan fractions are known, however, structural information is lacking. Recently, the major proteoglycan of this cartilage was identified as aggrecan. In this study, global molecular images and glycosaminoglycan structure of salmon nasal cartilage aggrecan purified from 4M guanidine hydrochloride extract were analyzed using HPLCs and atomic force microscopy with bovine tracheal cartilage aggrecan as a control. The estimated numbers of sulfates per disaccharide unit of chondroitin sulfate chains of salmon and bovine aggrecans were similar (approximately 0.85). However, the disaccharide composition showed a higher proportion of chondroitin 6-sulfate units in salmon aggrecan, 60%, compared to 40% in bovine. Gel filtration HPLC and monosaccharide analysis showed the salmon aggrecan had a lower number (approximately one-third), but 1.5–3.3 times longer chondroitin sulfate chains than the bovine aggrecan. Atomic force microscopic molecular images of aggrecan supported the images predicted by biochemical analyses.

Blood coagulation induced by Iranian saw-scaled viper (echis carinatus) venom: identification, purification and characterization of a prothrombin activator.

Echis carinatus is one of the venomous snakes in Iran. The venom of Iranian Echis carinatus is a rich source of protein with various factors affecting the plasma protein and blood coagulation factor. Some of these proteins exhibit types of enzymatic activities. However, other items are proteins with no enzymatic activity. In order to study the mechanism and effect of the venom on human plasma proteins, the present study has evaluated the effect of crude venom and all fractions. A procoagulant factor (prothrombin activator) was isolated from the venom of the Iranian snake Echis carinatus with a combination of gel filtration (Sephadex G-75), ion-exchange chromatography (DEAE- Sepharose) and reverse phase HPLC. Furthermore, proteolytic activity of the crude venom and all fractions on blood coagulation factors such as prothrombin time (PT) was studied. In the present study, the PT test was reduced from 13.4 s to 8.6 s when human plasma was treated with crude venom (concentraion of venom was 1 mg/ml). The purified procoagulant factor revealed a single protein band in SDS polyacrylamide electrophoresis under reducing conditions and its molecular weight was estimated at about 65 kDa. A single-band protein showed fragment patterns similar to those generated by the group A prothrombin activators, which convert prothrombin into meizothrombin independent of the prothrombinase complex. This study showed that the fraction which separated from Iranian snake Echis carinatus venom can be a prothrombin activators. It can be concluded that this fraction is a procoagulant factor.

Vietnamese Heterometrus laoticus scorpion venom: Evidence for analgesic and anti-inflammatory activity and isolation of new polypeptide toxin acting on Kv1.3 potassium channel

The scorpion Heterometrus laoticus (Scorpionidae) inhabits Indochinese peninsula and is widely distributed in South-West Vietnam. Since no human fatalities caused by H. laoticus stings were reported, no systematic characterization of the venom was earlier done. In this study we report on biological activity of the venom from H. laoticus caught in Vietnamese province An Giang. The venom manifested a very low acute toxicity with LD50 of about 190 mg/kg body weight in mice at subcutaneous (s.c.) injection and 12 mg/kg at intravenous injection. The venom analgesic effects using tail immersion and writhing tests as well as anti-inflammatory effect using carrageenan test were analyzed at doses of 9.5 and 19 mg/kg at s.c. injections. It was found that at two doses tested H. laoticus venom showed both anti-nociceptive and anti-inflammatory activity. The venom was fractionated by means of gel-filtration and reversed-phase HPLC. As a result several polypeptide toxins were isolated and new toxin hetlaxin was identified. Its amino acid sequence was determined and binding to the extracellular vestibule of the K+-conducting pore of Kv1.1 and Kv1.3 potassium channels was studied. Hetlaxin belongs to the scorpion alpha-toxin family and is the first toxin isolated from H. laoticus venom which possesses high affinity (Ki 59 nM) to Kv1.3 potassium channel.

Purification, kinetics, inhibitors and CD for recombinant β‐amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

β-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant β-amyrin synthase. The β-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni(2+) -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30 °C, respectively. The kinetic parameters, K(m) and k(cat) , were determined to be 33.8 ± 0.53 μm and 46.4 ± 0.68 min(-1), respectively. To the best of our knowledge, there are no reports describing both K(m) and k(cat) for OSCs except for two examples of rat and bovine lanosterol synthases. The β-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k(cat)/K(m)) (~ 10(3)-fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K(i) and IC(50) and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted for the DCTAE(485-489) motif, which is a putative initiation site for the polycyclization reaction. No activity of the D485N variant and significantly decreased activity of the C564A variant were found, definitively demonstrating that the acidic carboxyl residue Asp485 serves as a proton donor to initiate the polycyclization reaction, and that Cys564 is involved in hydrogen bond formation with the carboxyl residue Asp458 to enhance the acidity. The CD spectrum is the first to be reported for OSCs, and the CD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations.

Purification of arsenic-binding proteins in hamster plasma after oral administration of arsenite

To purify the arsenic-binding proteins (As-BP) in hamster plasma after a single oral administration of arsenite (iAs(III)). Arsenite was given to hamsters in a single dose. Three types of HPLC columns, size exclusion, gel filtration and anion exchange columns, combined with an inductively coupled argon plasma mass spectrometer (ICP MS) were used to purify the As-BP in hamster plasma. SDS-PAGE was used to confirm the arsenic-binding proteins at each purification step. The three-step purification process successfully separated As-BP from other proteins (ie, arsenic unbound proteins) in hamster plasma. The molecular mass of purified As-BP in plasma was approximately 40-50 kD on SDS-PAGE. The three-step purification method is a simple and fast approach to purify the As-BP in plasma samples.

Analysis of the Components of Porcine Liver Hydrolysate and Examination of the Antioxidant Activity and Angiotensin Converting Enzyme (ACE)-inhibiting Activity

Hypertension and oxidant stress predispose to the onset and progression of arteriosclerotic diseases. In this study, the components of two kinds of porcine liver hydrolysates (LH-I and LH-II) were analyzed, and the antioxidant effects and angiotensin converting enzyme (ACE)-inhibiting effects of LH-I and LH-II were examined in vitro. Furthermore, the effects of LH-I and LH-II on the blood pressure were examined in spontaneously hypertensive rats (SHR). The results showed that peptides and amino acids accounted for 70% or more of the constituents of both LH-I and LH-II. The results of gel filtration HPLC showed that most of the nitrogen-containing components were peptides or amino acids with molecular weights of 6000 or less. The DPPH radical scavenging activities of LH-I and LH-II were 55.6 and 38.1 μM Trolox Equivalent/g, respectively. The IC(50) values for the ACE-inhibiting activity of LH-I and LH-II were 0.18 and 0.31 mg/mL, respectively. Oral administration of 1 g/rat of LH-I or LH-II to SHR resulted in significant lowering of the blood pressure. These findings indicate that both LH-I and LH-II have antioxidant activity and ACE-inhibiting activity. Moreover, both exerted a blood pressure-lowering effect in SHR. The antioxidant activity and ACE-inhibiting activity of LH-I and LH-II are presumed to be based on the actions of the component peptides.

A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression

A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6nmol min(-1) mg(-1), and their optical purities were 92.7% ee, 96.4% ee, and >99% ee, respectively. Using a NADPH-regenerating system, 10mM 2-MPN was converted to amine with 100% conversion and 92% ee after 24h. The amino acid sequence analysis revealed that SIR showed about 60% identity to 6-phosphogluconate dehydrogenase. However, it showed only 37% identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.

Activation of Alpha Chymotrypsin by Three Phase Partitioning Is Accompanied by Aggregation

Precipitation of alpha chymotrypsin in the simultaneous presence of ammonium sulphate and t-butanol (three phase partitioning) resulted in preparations which showed self aggregation of the enzyme molecules. Precipitation with increasing amounts of ammonium sulphate led to increasing size of aggregates. While light scattering estimated the hydrodynamic diameter of these aggregates in the range of 242–1124 nm; Nanoparticle tracking analysis (NTA) gave the value as 130–462 nm. Scanning electron microscopy and gel filtration on Sephadex G-200 showed extensive aggregation in these preparations. Transmission electron microscopy showed that the aggregates had irregular shapes. All the aggregates had about 3× higher catalytic activity than the native enzyme. These aggregates did not differ in λmax of fluorescence emission which was around 340 nm. However, all the aggregates showed higher fluorescence emission intensity. Far-UV and near-UV circular dichroism also showed no significant structural changes as compared to the native molecule. Interestingly, HPLC gel filtration (on a hydroxylated silica column) gave 14 nm as the diameter for all preparations. Light scattering of preparations in the presence of 10% ethylene glycol also dissociated the aggregates to monomers of 14 nm. Both these results indicated that hydrophobic interactions were the driving force behind this aggregation. These results indicate: (1) Even without any major structural change, three phase partitioning led to protein molecules becoming highly prone to aggregation. (2) Different methods gave widely different estimates of sizes of aggregates. It was however possible to reconcile the data obtained with various approaches. (3) The nature of the gel filtration column is crucial and use of this technique for refolding and studying aggregation needs a rethink.

The N-terminal Capping Propensities of the D-helix Modulate the Allosteric Activation of the Escherichia coli cAMP Receptor Protein

Transduction of biological signals at the molecular level involves the activation and/or inhibition of allosteric proteins. In the transcription factor cAMP receptor protein (CRP) from Escherichia coli, the allosteric activation, or apo-holo transition, involves rigid body motions of domains and structural rearrangements within the hinge region connecting the cAMP- and DNA-binding domains. During this apo-holo transition, residue 138 is converted as part of the elongated D-helix to the position of the N-terminal capping residue of a shorter D-helix. The goal of the current study is to elucidate the role of residue 138 in modulating the allostery between cAMP and DNA binding. By systematically mutating residue 138, we found that mutants with higher N-terminal capping propensities lead to increased cooperativity of cAMP binding and a concomitant increase in affinity for lac-DNA. Furthermore, mutants with higher N-terminal capping propensity correlate with properties characteristic of holo-CRP, particularly, increase in protein structural dynamics. Overall, our results provide a quantitative characterization of the role of residue 138 in the isomerization equilibrium between the apo and holo forms of CRP, and in turn the thermodynamic underpin to the molecular model of allostery revealed by the high resolution structural studies.

Azemiopsin from Azemiops feae Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor

Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT(3) receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.