A simple one-step purification of human α1-proteinase inhibitor by immunoadsorbent column chromatography

A one-step purification of human α₁-proteinase inhibitor was described using the rabbit anti-α₁-proteinase inhibitor antibody coupled to CNBr-activated Sepharose 4B. The elution of α₁-proteinase inhibitor from the immunoadsorbent column using 0.1 M Na₂CO₃/0.5 M NaCl solution gave an 85% yield. The properties of eluted α₁-proteinase inhibitor were identical with that of α₁-proteinase inhibitor that was purified by the conventional method. In addition, the specific activity of purified α₁-proteinase inhibitor was more than 93% of that of the theoretical value.     

Binding of a Phosphorylated Inhibitor to Ribulose Bisphosphate Carboxylase/Oxygenase during the Night

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured at various times during the purification of the enzyme from leaves of Nicotiana tabacum which were collected either 1 hour before the start of the photoperiod (predawn) or in the middle of the photoperiod (midday). The activity of the enzyme in extracts of the predawn leaves (0.8 units/mg enzyme) was consistently about 2-fold lower than that measured in extracts of midday leaves (1.7 units/mg enzyme). The activity of the predawn enzyme was increased to that of the midday enzyme following removal of CO₂ and Mg²⁺ (deactivation), (NH₄)₂SO₄ precipitation, or incubation in SO₄²⁻ (18 millimolar required for one-half maximal increase). Following purification to >95% homogeneity, the predawn enzyme was found to have ~0.5 moles of bound organic phosphate per mole of enzyme active sites, while the midday enzyme had only ~0.08 moles of bound organic phosphate per mole of enzyme active sites. Deactivation of the predawn enzyme or treatment with 0.2 molar SO₄²⁻ resulted in the removal of most of the bound organic phosphate. These findings support the hypothesis that following the night period about 50% of the enzyme is catalytically inactive because of the tight-binding of a small molecular weight, phosphorylated inhibitor at the active site.     

Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems

Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH₄)₂SO₄], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH₄)₂SO₄ system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH₄)₂SO₄ and 1.0?mol l^?1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.     

Threonine-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum

Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg²⁺ or Mn²⁺, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and K_i for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine.     

Regulation of Rubisco by inhibitors in the light

2-carboxy-D-arabinitol-1-phosphate (CA1P) bound to Rubisco either in leaf extracts or after purification can be displaced by SO₄^2? ions. Thus, treatment of leaf extracts with a buffer containing 200 mol m^?3 SO₄^2? displaces any bound CA1P and enables measurement of maximum car-boxylation potential. In tobacco leaves, the activity following treatment with SO₄^?2 ions (‘maximal activity’) is greater than the total Rubisco activity. The ratio of the two activities altered in a dynamic way with fluctuations in irradiance. Even in species which do not produce significant amounts of CA1P, the maximal activity greatly exceeded the total activity. Anion exchange separation of components in acid extracts confirmed the absence of CA1P in tobacco leaves harvested above an irradiance of 300 μmol quanta m^?2 s^?1, but the presence of another inhibitor of Rubisco. These results are consistent with the regulation of Rubisco activity by inhibitors other than CA1P which, like CA1P, can be displaced by SO₄^2? ions.     

Purification of Proteins by the Use of Hydrophobic Zeolite Y

Hydrophobic zeolite Y can be used as a fast and efficient and inexpensive matrix in the purification of proteins from crude extracts. Preferably the zeolite can be used in the first purification step, replacing the commonly used precipitation techniques with (NH₄)₂SO₄ or ethanol. The time required for the zeolite prefractionation was a few hours compared to the much more time consuming precipitation procedure which demands centrifugation and subsequent dialysis. Proteins can be adsorbed on the zeolite either in order to remove undesired proteins or to be subsequently eluted from the zeolite in order to achieve purification and concentration. Removal of undesired proteins is exemplified by the purification of horseradish peroxidase from a crude extract. The zeolite procedure enhanced the specific activity five times and provided a yield similar to that which was obtained by the use of standard procedures, (NH₄)₂SO₄ fractionation and ion-exchange chromatography. Binding and subsequent elution of proteins from the zeolite is exemplified by the purification of monoclonal antibodies from hybridoma culture supernatants. Proteins were desorbed from the zeolite by the use of polyethylene glycol 600 and this procedure yielded a purification factor of 5.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

利用ELP-Intein系统在大肠杆菌中生产抗菌肽DLP4

DLP4 （defensin-like peptide 4）是一种新型昆虫防御素抗菌肽,对革兰氏阳性细菌具有强大的抗菌活性而且不易产生抗药性。本研究利用类弹性蛋白（elastin-like polypeptide, ELP）的相变特性和蛋白质内含子（intein, I）的C端断裂系统,通过构建重组表达质粒pET-ELP-I-DLP4,以大肠杆菌（Escherichia coli）作为宿主细胞,诱导表达后的重组蛋白通过简单的离心、pH和温度转变进行纯化得到DLP4。研究中发现,在表达纯化过程中蛋白质内含子发生了C端提前断裂。为了解决这一问题,将其断裂为N端片段（I0_N）和C端片段（I0_C）后,分别与ELP或DLP4融合,构建了pET-ELP-I0_N和pET-ELP-I0_C-DLP4两种重组表达质粒。分别在大肠杆菌中诱导表达,将表达后的菌液混合,使蛋白质内含子恢复C端断裂活性,最终得到的DLP4的得率约为1.49 mg/L。抑菌试验证明纯化的DLP4表现出预期活性,这为DLP4在原核系统中的表达纯化提供...     

Advances in the Purification of the Mitochondrial Ca2+ Uniporter Using the Labeled Inhibitor 103Ru360

For many years the calcium uniporter has eluded attempts of purification, partly because of the difficulties inherent in the purification of low-abundance hydrophobic proteins (Reed and Bygrave, 1974). Liquid-phase preparative isoelectric focusing improved the fractionation of mitochondrial membrane proteins. A single 6-h run resulted in a 90-fold increase in specific activity of pooled active fractions over a semipurified fraction, allowing for enrichment of the calcium transport function in cytochrome oxidase vesicles. An additional powerful tool in the isolation of the uniporter was the use of the labeled inhibitor ¹⁰³Ru₃₆₀ as an affinity ligand; by following this procedure a protein of 18 kDa was purified in nondenatured, but rather inactive, form. The labeled protein corresponds to the protein that showed Ca²⁺ transport activity.     

Evaluation of immobilized metal affinity chromatography for purification of penicillin acylase

The aim of this work was to test immobilized metal affinity chromatography (IMAC) for the purification of penicillin acylase. After evaluation of different metals, Cu²⁺ was selected. Different samples were tested: pure penicillin acylase, industrial clarified feedstock and crude extract. After comparing two eluents, NH₄Cl and imidazole, it appeared that although both gave good results for recovery and activity, NH₄Cl was a more selective eluent with a higher fold purification than imidazole (4.64 versus 2.04). Moreover, we shown that a multistep gradient of NH₄Cl, greatly increased the degree of purification (12.36) compared with the one-step process as control (4.64). In addition, good recovery was obtained (97–100%).     

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)1

Enolase from Synechococcus PCC 6301 was purified 1450‐fold to electrophoretic homogeneity and a final specific activity of 68 μmol of phosphoenolpyruvate produced·min^?1·mg protein^?1. Analytical gel filtration and nondenaturing and SDS‐gel electrophoresis demonstrated that this enolase exists as a 118‐kDa homodimer composed of 56‐kDa subunits. The purified enzyme displayed 1) a broad pH‐activity profile with maximal activity occurring at pH 8.0 and 7.5 for the forward and reverse reactions, respectively, 2) a forward‐to‐reverse maximal activity ratio of about 1.6, 3) a K_m (2‐phosphoglycerate) of 0.28 mM, and 4) an absolute requirement for a divalent metal cation cofactor that was best satisfied by Mg²⁺ (K_m=0.62 mM). Enolase activity increased by about 200% after the first purification step (60° C heat treatment), whereas addition of increasing amounts of a clarified extract led to a progressive 70% inhibition in the activity of the purified enzyme. This was reflected by a reduction in enolase's V_max from 73 to 22 U·mg^?1 and forward‐to‐reverse activity ratio from 1.6 to 1.3. This inhibition was negated when the clarified extract was either preincubated with trypsin or warmed to approximately 40° for 5 min. Results are indicative of a heat‐labile enolase inhibitor protein in Synechococcus PCC 6301. By contrast, the purified enolase lost no activity when incubated at 70° C for up to 5 min. This study represents the first purification of enolase from the Cyanophyceae. Characterization of the purified enzyme's physical and kinetic features has provided insights into the structural and functional properties of cyanobacterial enolase.     

In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases

The inhibition of two human carbonic anhydrase (HCA, EC 4.2.1.1) isozymes, the cytosolic HCA I and II, with heavy metal salts of Pb(II), Co(II) and Hg(II)has been investigated. Human erythrocyte CA-I isozyme was purified with a specific activity of 920 EUmg^{? 1} and a yield of 30% and CA-II isozyme was purified with a specific activity of 8000 EUmg^{? 1} and a yield of 40% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 104-fold for HCA-I and 900-fold for HCA-II. The inhibitory effects of different heavy metals (lead, cobalt and mercury) on CA activity were determined at low concentrations using the esterase method under in vitro conditions. K_i values for these metals were calculated from Lineweaver-Burk graphs as 1.0, 3.22 and 1.45 mM for HCA-I and 0.059, 1.382 and 0.32 mM for HCA-II respectively. Lead was a noncompetitive inhibitor for HCA-I and competitive for HCA-II, cobalt was competitive for HCA-I and noncompetitive for HCA-II and mercury was uncompetitive for both HCA-I and HCA-II. Lead was the best inhibitor for both HCA-I and HCA-II.     

Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate

A two-step chromatographic purification procedure from clarified Escherichia coli ultrasonic homogenate was evaluated. The capture step included immobilized metal affinity chromatography with Cu²⁺ as metal ion. Two elution methods were performed: 1 M NH₄Cl and 0.01 M imidazole. Respectively, we obtained a different purification fold (16.5 to 3.15) and a similar result for the recovery of activity (90–99%). The best elution method was chosen for the procedure. The second step, hydrophobic interaction chromatography, gave a 3.8-fold purification with 77.7% of activity. The total procedure gave a 66-fold purification in relation to the initial crude extract with 70% for the recovery of activity and was performed without any conditioning step and at the same pH value.     

Extraction and three-phase partitioning behavior of proteases from papaya peels

Dried papaya peels exhibited superior proteolytic activity to the fresh peels. An extraction with phosphate buffer pH 7.0 greatly maintained proteolytic activity when compared to water. SDS-PAGE verified that the extracted dried papaya peels held a wide range of proteins. To optimize the three-phase partitioning (TPP) for isolating the papaya peel proteases required a ratio of crude extract to t-butanol, the (NH₄)₂SO₄ concentration and the TPP cycles. The ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 20% (NH₄)₂SO₄ resulted in the highest proteolytic recovery at 253.5%, and 15.8-fold of purification in the bottom phase. The TPP was then optimized by adding up to 55% (NH₄)₂SO₄ to the bottom phase of the first step. A purification of 10.1-fold with about 89.4% recovery was obtained. This study showed the TPP can be effectively employed for the extraction of proteases from papaya peels.     

牛小脑肌醇磷脂激酶PI(4)K高产率纯化与特征

对牛小脑膜区肌醇磷脂激酶进行了11 500倍纯化,过程包括：TritonX-100抽提,硫酸铵沉淀,阳离子交换层析(phosphocellulose),亲和层析(Heparin Sepharose CL-6B)和阴离子交换层析(DEAE10,FPLC)等.纯化程度可达95％以上,对SDS-PAGE电泳结果进行扫描分析测其分子质量为56 ku.纯化的肌醇磷脂激酶的特异活性为450 nmol/mg·min, 动力学性质表现为ATP的表观K_m值为7.9×10^-7 mol/L,PI的表观K_m值为6.6×10^-7 mol/L. 腺嘌呤核苷是该酶的有效抑制剂,3.5×10^-7 mol/L腺嘌呤核苷可使该酶活力降低约50％,而TritonX-100对该酶活力具有刺激作用,0.5% TritonX-100可使该酶表现为最高活力.     

Partial purification and properties of a multifunctional 3′,5′-cyclic nucleotide phosphodiesterase from Lactuca cotyledons

The extraction, partial purification and properties of a 3′,5′-cyclic nucleotide phosphodiesterase from lettuce cotyledons is described. Purification involved fractional precipation with (NH₄)₂SO₄, chromatography on Sephadex G-200, affinity chromatography on Affi-Gel Blue and non-denaturing polyacrylamide gel electrophoresis. The behaviour of the final enzyme preparation on SDS-polyacrylamide gel electrophoresis was examined and inidcated an M, of ca 62 000. The enzyme from 3′,5′-cyclic nucleotide phosphodiesterases previously isolated from plant tissues in that it exhibits activity towards pyrimidine as well as purine cyclic nucleotides. Furthermore, it hydrolyses cyclic CMP at a comparable rate to that with which it hydrolyses cyclic AMP and cyclic GMP. Both 3′- and 5′-AMP were released, with the 5′-nucleotide being the major product. Whereas the K_m with all three substrates remained constant during the purification procedure, V_max with cyclic AMP was lower than that for cyclic CMP but increased as purification proceeded. The effects were examined of a range of di- and trivalent metal ions on the enzyme activity. Fe³⁺ significantly stimulated the activity, more so when cyclic GMP was the substrate. Cu²⁺ inhibited the activity.     

Separation of proteins and viruses using two-phase aqueous micellar systems

We discuss the utilization of a novel two-phase aqueous nonionic micellar system for the purification and concentration of biomolecules, such as proteins and viruses, by liquid–liquid extraction. The nonionic surfactant n-decyl tetra(ethylene oxide), C₁₀E₄, phase separates in water into two coexisting aqueous micellar phases by increasing temperature. The mild interactions of the C₁₀E₄ nonionic surfactant with biomolecules, combined with the high water content of the two coexisting micellar phases, suggest the potential utility of two-phase aqueous C₁₀E₄ micellar systems for the purification and concentration of biomolecules. In this paper, we review our recent experimental and theoretical studies involving the partitioning of several water-soluble proteins, including cytochrome c, soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and catalase, in the two-phase aqueous C₁₀E₄ micellar system. In addition, we present results of our preliminary experimental investigation on the partitioning of bacteriophages, including φX174, P22, and T4.     

Identification of a potent xanthine oxidase inhibitor from oxidation of caffeic acid

Inhibitory activity of Fe-ion-catalyzed radical oxidation products from 22 types of phenolic compounds toward xanthine oxidase (XO) was investigated. Phenols are readily oxidizable compounds in nature and, thus, showed potent antioxidant activities. Among the phenols screened in this study, noticeable activity was observed in the oxidation product of caffeic acid, whereas almost no XO-inhibitory activity of caffeic acid was observed. Assay-guided purification of the oxidation product of caffeic acid afforded a highly potent XO inhibitor, with an IC₅₀ value that was calculated to be 60 nmol L⁻¹, which indicated XO-inhibitory activity much stronger than that of allopurinol (IC₅₀ = 1 μmol L⁻¹), a potent XO inhibitor and excellent medicine for the treatment of gout. The chemical structure of this new XO inhibitor was investigated by one- and two-dimensional NMR and HR–ESI–MS analyses, and the unique tetracyclic structure was confirmed by synthesis starting from commercially available 1,2,4-trimethoxybenzene and 3,4-dimethoxylbenzoyl chloride.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Purification,characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek) seeds

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH₄(SO₄)₂ fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.