Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

Improved HPLC method for the simultaneous determination of tramadol and O-desmethyltramadol in human plasma

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.     

A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.     

Direct electron transfer of redox proteins at the bare glassy carbon electrode

A simple system is presented for the microscale, direct voltammetry of redox proteins, typically 25 micrograms, in the absence of mediators and/or modifiers. The sample consists of a droplet of anaerobic solution laid onto an oversized disc of nitric-acid-pretreated glassy carbon as the working electrode. Very reproducible, Nernstian responses are obtained with horse heart cytochrome c. The midpoint potential Em (pH 7.0) is dependent on the ionic strength, ranging from $293 mV in 1 mM potassium phosphate to $266 mV in 0.1 M phosphate. At fixed buffer and cytochrome concentrations the magnitude of the voltammetric response is found to be independent of pH over six pH units around neutrality. It is suggested that the response of the present system is not complicated by pH-dependent properties of the electrode surface around physiological pH and, therefore, that the use of this system is practical in biochemically oriented studies. Direct, quasi-reversible responses have also been obtained at pH 7.0 (5 mM phosphate) from Desulfovibrio vulgaris. Hildenborough strain, tetraheme cytochrome c3 (pI = 10.0 at 4 C; 3 X Em = -0.32 mV, Em = -0.26 V), and cytochrome c553 (pI = 9.3; Em = +60 mV), and from Megasphaera elsdenii rubredoxin (pI congruent to 3; Em = -353 mV). The latter protein absorbs onto the glassy carbon surface, thus forming a system with possible applications in the electrochemical study of ferredoxin-linked enzymes.     

Hypertonic sodium bicarbonate partially reverses QRS prolongation due to flecainide in rats

Hypertonic (1M) sodium bicarbonate can partially reverse the cardiac toxicity of some Class IA antiarrhythmic agents, presumably by antagonizing sodium channel inhibition. We studied the effects of 1M sodium bicarbonate on toxicity due to the Class IC drug flecainide. Anesthetized rats received i.v. loading and maintenance doses of flecainide to produce QRS prolongation of 76% that was stable over the 60 min study period. 20 min after the start of the maintenance infusion, groups of 8 rats received an i.v. infusion of 1M sodium bicarbonate (6 meq/kg) or an equal volume of 0.9% saline. QRS prolongation was reduced by 1M sodium bicarbonate but not only 0.9% saline (% change -12.2 +/- 10.0 v. +3.0 +/- 2.7, p = 0.001). Expressed as a percent of the flecainide-induced QRS prolongation, bicarbonate reduced this prolongation by 26.5%. The improvement in QRS duration persisted until sacrifice 30 min later. Compared to controls, the bicarbonate group had a significantly higher blood pH (7.55 +/- 0.06 v. 7.44 +/- 0.05) and serum sodium concentration (149 +/- 5 v. 137 +/- 2 meq/l). Serum flecainide concentrations were similar. These data suggest that 1M sodium bicarbonate can partially reverse flecainide-induced conduction delay in rats. This effect may be due to changes in the extracellular pH and sodium concentration. 1M sodium bicarbonate may be useful in assessing the role of sodium channel inhibition in mediating the toxicity of flecainide or other Class IC drugs.     

Prevention of aggregation after refolding by balanced stabilization-destabilization: production of the Arabidopsis thaliana protein APG8a (At4g21980) for NMR structure determination

The gene coding for APG8a (At4g21980), a protein from Arabidopsis thaliana, is involved in the autophagy process. The protein is an interesting candidate for structure determination by NMR spectroscopy. Toward this end, APG8a fused to an N-terminal His-tag has been expressed in Escherichia coli under a T7 expression system, refolded in vitro, and kept soluble by slight destabilization. The expressed protein appeared in both the soluble and the insoluble fractions. The whole-cell lysate was denatured by the addition of guanidinium chloride. The protein was immobilized on nickel-agarose resin and refolded by stepwise decrement of the denaturant. The elution buffer was 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, 300 mM NaCl, and 1 M imidazole. After the removal of imidazole by ultrafiltration, the His-tag was cleaved with biotinylated thrombin. The protein product was kept in 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, and 300 mM NaCl. The protein was found to aggregate extensively over time if any one of the three ingredients (sodium chloride, urea, or glycerol) was omitted. The yield of the protein was around 20 mg/L Luria-Bertani culture medium. The (1)H-(15)N NMR correlation spectrum of (15)N-labeled APG8a showed the characteristic signature of a folded protein; thus, the solutes appear to have no deleterious effect on the sample. These solution conditions kept the protein soluble and unaggregated for at least 2 days (enough time for NMR data collection). This approach of balanced stabilization-destabilization may offer a general approach for structural investigations of proteins that tend to aggregate.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Crystallization of a complex between ribonuclease T1 and 2'-guanylic acid

Ribonuclease T1 was crystallized under various conditions. Form I crystals were produced by microdialysis against 53% (v/v) 2-methyl-2,4-pentanediol in 0.01 M sodium acetate, 0.05% 2'-guanylic acid (2'GMP) and 0.02% NaN3 (pH 6.2-7.2). These crystals are tetragonal, space group P41212 and contain two molecules per asymmetric unit; cell dimensions are a = b = 5.86 nm, c = 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01-0.05 M sodium acetate, 0.25-0.5% 2'GMP, 0.02% NaN3 and 2-5 mM calcium acetate (pH 4.0-4.4) in the presence of 50-75% (v/v) 2-methyl-2,4-pentanediol. These crystals are orthorhombic, space group P212121, and contain one molecule per asymmetric unit; cell dimensions are a = 4.66 nm, b = 5.02 nm, c = 4.04 nm (form I) and alpha = 4.44 nm, b = 5.00 nm, c = 4.03 nm (form II). Using high-performance liquid chromatography, it could be shown for all crystal forms that 2'-GMP is bound in the crystals. The molecular ratio between RNase T1 and 2'GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme-nucleotide complex. Heavy-atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three-dimensional X-ray analysis of the RNase-T1 x 2'GMP complex is under way.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose.

Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested.     

Process optimization for reverse micellar extraction of stem bromelain with a focus on back extraction

In the current study, reverse micellar extraction (RME) for the purification of stem bromelain was successfully achieved using the sodium bis(2‐ethylhexyl) sulfosuccinate (AOT)/isooctane system. A maximum forward extraction efficiency of 58.0% was obtained at 100 mM AOT concentration, aqueous phase pH of 8.0 and 0.2 M NaCl. Back extraction studies on altering stripping phase pH and KCl concentration, addition of counter‐ion and iso‐propyl alcohol (IPA) and mechanical agitation with glass beads indicated that IPA addition and agitation with glass beads have significant effects on extraction efficiency. The protein extraction was higher (51.9%) in case of the IPA (10% v/v) added system during back extraction as compared to a cetyltrimethylammonium bromide (100 mM) added system (9.42%). The central composite design technique was used to optimize the back extraction conditions further. Concentration of IPA, amount of glass beads, mixing time, and agitation speed (in rpm) were the variables selected. IPA concentration of 8.5% (v/v), glass bead concentration of 0.6 (w/v), and mixing time of 45 min at 400 rpm resulted in higher back extraction efficiency of 45.6% and activity recovery of 88.8% with purification of 3.04‐fold. The study indicated that mechanical agitation using glass beads could be used for destabilizing the reverse micelles and release of bromelain back into the fresh aqueous phase. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:845–855, 2014     

Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing

Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of previously unreported membrane proteins.     

Hydrolysis of fucosyl-GM1 ganglioside by purified pellet-associated human brain and human liver alpha-L-fucosidases without activator proteins or detergents.

Pellet-associated human brain alpha-L-fucosidase was solubilized with 0.5% (w/v) Triton X-100 and purified by affinity chromatography on agarose-6-aminohexanoyl-fucosamine resin. The procedure resulted in a 290,000-fold purification, a 58% yield and a final specific activity of 11,500 nmol/min per mg of protein. Isoelectric focusing indicated that all six major isoforms (with pI values between 4.1 and 5.3) present in crude brain pellet preparations were purified by the affinity technique. SDS/PAGE indicated the presence of one subunit (54 kDa) and a minor protein band at 67 kDa, which presumably is a contaminant since it was not immunoreactive on Western blotting. The pH optimum of the brain enzyme and its apparent Km for the synthetic substrate 4-methylumbelliferyl alpha-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver alpha-L-fucosidases were both capable of hydrolysing fucosyl-GM1 ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37 degrees C was found at pH 3.4 for both alpha-L-fucosidases, with no activity at pH values above 4.0.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

以苦瓜籽为材料,研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明,柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时,各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点,pH6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液(pH4.0),1%PAA选择性沉淀碱性蛋白(等电点pI为8.65~9.30)的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0,等电点沉淀后的上清液用PAA沉淀碱性蛋白,当PAA(1%)用量为160μL/mL提取液时,pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀;当PAA用量为120μL/mL提取液时,pH4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液(pH>9.0),当溶液NaCl浓度为3.0%时,溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经SephadexG-75柱层析分离,分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD,pI值约为9.5,主峰Ⅱ的分子量约为10kD,pI值约为9.3。     

Purification by Immunoaffinity Chromatography, Characterization, and Structural Analysis of a Thermostable Pyranose Oxidase from the White Rot Fungus Phlebiopsis gigantea

A moderately thermostable pyranose oxidase (PROD) was purified to apparent homogeneity with a yield of 71% from mycelium extracts of the white rot fungus Phlebiopsis gigantea by an efficient three-step procedure that included heat treatment, immunoaffinity chromatography, and gel filtration on Superdex 200. PROD of P. gigantea is a glycoprotein with a pI between pH 5.3 and 5.7. The relative molecular weight (M(infr)) of native PROD is 295,600 (plusmn) 5% as determined by four independent methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PROD revealed two distinct but similar stained bands corresponding to polypeptides with M(infr)s of 77,000 and 70,000, suggesting a heterotetrameric enzyme structure. The tetrameric structure of PROD was confirmed by electron microscopic examinations, which additionally showed the ellipsoidal shape (4.6 by 10 nm) of each subunit. Spectral analyses and direct determinations showed the presence of covalently bound flavin adenine dinucleotide with a stoichiometry of 3.12 mol/mol of enzyme. A broad pH optimum was determined in the range pH 5.0 to 8.0 in 100 mM sodium phosphate, and the activation energy for d-glucose oxidation was 24.7 kJ/mol. The main substrates of PROD are d-glucose, l-sorbose, and d-xylose, for which K(infm) values 1.2, 16.5, and 22.2 mM were determined, respectively. PROD showed high stability during storage. In 100 mM sodium phosphate (pH 6.0 to 8.0), the half-life of PROD activity was >300 days at 40(deg)C, >110 days at 50(deg)C (pH 7.0), and 1 h at 65(deg)C.     

Determination of atorvastatin in human serum by reversed-phase high-performance liquid chromatography with UV detection

A rapid and sensitive high-performance liquid chromatographic method was validated and described for determination of atorvastatin in human serum. Following liquid-liquid extraction of the drug and an internal standard (sodium diclofenac), chromatographic separation was accomplished using C18 analytical column with a mobile phase consisting of sodium phosphate buffer (0.05 M, pH 4.0) and methanol (33:67, v/v). Atorvastatin and the internal standard were detected by ultraviolet absorbance at 247 nm. The average recoveries of the drug and internal standard were 95 and 80%, respectively. The lower limits of detection and quantification were 1 and 4 ng/ml, respectively, and the calibration curves were linear over a concentration range of 4-256 ng/ml of atorvastatin in human serum. The analysis performance was studied and the method was applied in a randomized cross-over bioequivalence study of two different atorvastatin preparations in 12 healthy volunteers.     

Chromatographic behavior of cyclic AMP dependent protein kinase and its subunits from Dictyostelium discoideum

An adenosine cyclic 3',5'-monophosphate (cAMP) dependent protein kinase has recently been shown to exist in Dictyostelium discoideum and to be developmentally regulated. In this report we have followed the chromatographic behavior of both the holoenzyme and its subunits. A cAMP-dependent holoenzyme could be obtained from the 100000 g soluble fraction after passage through DE-52 cellulose (pH 7.5) and Sephacryl S300. Under conditions of low pH the holoenzyme could be further purified by flat-bed electrofocusing (pI = 6.8). Application of the holoenzyme to electrofocusing at high pH resulted in dissociation of the holoenzyme into a cAMP binding component (pI = 6.1) and a cAMP-independent catalytic activity (pI = 7.4). Dissociation of the holoenzyme into subunits also occurred during histone affinity chromatography and gel filtration chromatography (S300) in the presence of a dissociating buffer. Although the subunit structure was clearly evident during chromatography, the holoenzyme could not be dissociated by simple addition of cAMP to the extract. The catalytic subunit could be purified further by CM-Sephadex, DE-52 cellulose (pH 8.5), histone affinity, and hydrophobic chromatography. The regulatory subunit was further purified by DE-52 cellulose (pH 8.5) and cAMP affinity chromatography. Proof that the cAMP binding activity and the cAMP-independent catalytic activity were in fact the regulatory and catalytic subunits was shown by reconstitution of the cAMP-dependent holoenzyme from the purified subunits. By using these separation procedures, one can obtain from extracts of Dictyostelium the subunits that are free of each other as well as free of any endogenous protein substrates.     

Ovostatin: a novel proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties, and tissue distribution of ovostatin

A proteinase inhibitor which has strong anti-collagenase activity was found in chicken egg white. The inhibitor (pI = 4.9) was purified by poly(ethylene glycol) (5.5-10%) precipitation and chromatography on Ultrogel AcA 34, DEAE-cellulose, and Sephacryl S-300. The final product was homogeneous on 5% polyacrylamide gel electrophoresis. Stoichiometric inhibition was observed with the inhibitor and rabbit synovial collagenase and thermolysin (1:1 molar ratio with thermolysin). The inhibitor ran on sodium dodecyl sulfate-gel electrophoresis with reduction as a single protein band of Mr = 165,000. The molecular weight of the native inhibitor was estimated to be 780,000 by sedimentation equilibrium centrifugation. Centrifugation analysis in 6 M guanidine hydrochloride and of the reduced sample gave M omega = 380,000 and M omega = 195,000, respectively, where M omega is the weight-average molecular weight determined by equilibrium ultra-centrifugation. The results indicated that the inhibitor molecule is a tetramer of identical subunits linked in pairs by disulfide bonds. Since the molecular weight and the quaternary structure of the inhibitor were similar to those of alpha 2-macroglobulin (alpha 2M) in plasma, chicken alpha 2M was isolated and compared with the inhibitor. The inhibitor was not sensitive to methylamine, whereas chicken alpha 2M was. No immunocross-reactivity was observed between the inhibitor and chicken alpha 2M. The NH2-terminal sequence of the egg white inhibitor is Lys-Glu-Pro-Glu-Pro-Gln-Tyr-Val-Leu-Met-Val-Pro-Ala. The sequence of chicken alpha 2M is Ser-Thr-Val-Thr-Glu-Pro-Gln-Tyr-Met-Val-Leu-Leu-Pro-Phe. Considerable homology was found between the two sequences and to the NH2-terminal sequence of human alpha 2M. Monospecific antibody raised against the egg white inhibitor was employed to examine the tissue distribution of the inhibitor. The inhibitor was found only in oviduct and egg white, but not in other tissues or serum of chickens.     

Affinities of various nucleases to DNA-Sepharose under non-digestive conditions: survey for productive affinity chromatography

1. It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions. In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied. 2. All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA. The non-digestive conditions were as follows. i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0. At the respective pH's, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose. Although S1 and P1 were anionic, they still adsorbed to the column. 3. All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH. The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1. The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength. On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.     

Characterization of alkaline proteases from a novel alkali-tolerant bacterium Bacillus patagoniensis

Summary The extracellular proteolytic activity produced by a moderately alkaliphilic bacterium, Bacillus patagoniensis PAT 05^T, was characterized. This strain, grown in a highly alkaline and saline medium, produced important levels of proteolytic activity. SDS-PAGE and zymogram analyses revealed two proteolytic active bands. Through isoelectricfocusing (IEF)-zymogram, an active band with alkaline pI and two slighter active bands with acid pI values were detected. The alkaline active enzyme in the IEF was purified and characterized. It showed a molecular mass of 29.4 kDa and its pI value was >‰10.3. Proteolytic activity of the culture supernatant showed an optimal temperature of approximately 60 °C and a plateau of maximum activity between pH 9.0 and 12.0. Such activity was not affected by H₂O₂ (10% v/v), 1,10-phenanthroline (10 mM), Triton X-100 (1% v/v) and Tween 20 (1% v/v), under the assay conditions. More than 80% of the activity was retained in 10 mM EDTA, 73% in 1 % (w/v) SDS and 63% in 2 M NaCl. The enzyme was inhibited by PMSF, indicating serine-protease activity. The proteolytic activity of the crude supernatant was thermosensitive with a half-life of 2.3 min at 70 °C, while high activity was detected at moderate temperatures. Considering PAT 05^T proteolytic activity characteristics, such as high optimum pH, high stability and residual activity in presence of oxidant, surfactant and chelating agents, this strain could be a potential source of enzymes for use as additives in detergent formulations or in the leather industry.