Separation of targeted ganoderic acids from Ganoderma lucidum by reversed phase liquid chromatography with ultraviolet and mass spectrometry detections

Ganoderic acids are valuable bioactive secondary metabolites produced by a traditional medicinal mushroom Ganoderma lucidum (“Ling-zhi” in Chinese and “Reishi” in Japanese). In this work, a fast and efficient method for the recovery and purification of ganoderic acid T (GA-T) and ganoderic acid Me (GA-Me) from triterpene-enriched extracts of G. lucidum mycelia was developed by using reversed phase HPLC (RP-HPLC) on a C₁₈ column with an acidified methanol–water mobile phase in combination with ultraviolet (UV) detection and electrospray ionization mass spectrometry (ESI-MS). The presence of each targeted GA (GA-T and GA-Me) in its corresponding peak was easily identified and confirmed by UV and MS. The chemical structures of the purified GA-T and GA-Me were further confirmed by ¹H NMR. The retention behaviors of the two GAs over a temperature range of 15–55 °C were also investigated. From the retention time data, van’t Hoff plots were obtained. The estimated enthalpy (ΔH) and entropy (ΔS) data suggest that the retention time difference between GA-T and GA-Me might be driven by an enthalpy difference. Furthermore, a semi-preparative HPLC purification was achieved on a semi-preparative C₁₈ column using the conditions optimized for the analytical column. The method presented in this work can be a valuable tool for the rapid semi-preparative purification of targeted GAs, and it may also be applicable to some other natural products.     

Simultaneous determination of hydroxocobalamin and its cyanide complex cyanocobalamin in human plasma by high-performance liquid chromatography application to pharmacokinetic studies after high-dose hydroxocobalamin as an antidote for severe cyanide poisoning

Hydroxocobalamin (OHCbl) is a powerful antidote for cyanide poisoning, via the formation of non-toxic cyanocobalamin (CNCbl). Plasmatic cobalamins were measured at 361 nm, after enrichment and purification on a short C₁₈ precolumn (1% acetic acid; 1 ml min⁻¹; 2 min), by back-flush elution on a C₁₈ ODS-2 column [0.1 M sodium dihydrogenphosphate-methanol (63:27, v/v) (pH 4.0); 0.80 ml min⁻¹]. The precision was 3.21 and 3.54% for 10 μM OHCbl and CNCbl, respectively. The method was used to study the pharmacokinetics of OHCbl and the formed CNCbl in severely poisoned patients.     

Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media

Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (q_max) of Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate for the GFP was 1,42,860 FU ml⁻¹ and 18,000 FU ml⁻¹, respectively. The expanded bed column chromatography using Ni²⁺ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni²⁺ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.     

Determination of diadenosine tetraphosphate (Ap4A) levels in subpicomole quantities by a phosphodiesterase luciferin-luciferase coupled assay: Application as a specific assay for diadenosine tetraphosphatase

A coupled enzymatic assay for diadenosine 5′, 5?-P¹, P⁴-tetraphosphate(Ap₄A) is described. Luciferin-luciferase produces light by consuming the ATP that is liberated by the action of snake venom phosphodiesterase on Ap₄A. The procedure is linear with Ap₄A levels ranging from 0.02 to 2 pmol. The pool size of Ap₄A in human leukemic cells was determined by acid extraction of the cells followed by initial fractionation of the extract on a DEAE-cellulose column and application of the phosphodiesterase luciferin-luciferase coupled assay. The method was also used to follow the purification of a diadenosine tetraphosphate-degrading enzyme (diadenosine tetraphosphatase, Ap₄Aase) from mouse ascites tumor cells. The partially purified enzyme had a K_m of 2.8 μm for Ap₄A when applying the coupled enzymatic assay for the determination of initial rate kinetics.     

Donut-shaped “miniparticles” in nuclei of human and rat cells

Donut-shaped “miniparticles” were extracted from nuclei of various types of human and rat cells. Electron-microscopic investigations showed these particles were predominantly in sucrose density gradient fractions that had an approximate sedimentation coefficient of 21S. These particles were 113±8_A^o in diameter and had an electron dense center of 29±6_A^o. They appeared to be composed of 8 subunits. Quantitative analysis of the number of these particles by electron-micrographic field counting showed nuclei of tumor samples had a larger amount of the particles than the cytosol. However, normal cell cytosol had a larger number of particles than the nuclei. A group of proteins in the 25, 000–33, 000 molecular weight range was shown to be the main protein component by two dimensional gel electrophoresis.     

Glucofructosan metabolism in Cichorium intybus roots

A 10-fold purification of sucrose sucrose fructosyl transferase from Cichorium intybus roots was achieved by ammonium sulphate fractionation and DEAE-cellulose column chromatography. The energy of activation for this enzyme was ca 48 kJ/mol sucrose. Sucrose sucrose fructosyl transferase and invertase were prominent during early months of growth. Evidence obtained from: (1) the changes in carbohydrate composition at monthly intervals; (2) comparative studies on fructosyl transferase and invertase at different stages of root growth; and (3) incubation studies with [¹⁴C]glucose, [¹⁴C]fructose and [¹⁴C]sucrose revealed that, during the later stages of root growth, fructosan hydrolase is responsible for fructosan hydrolysis. No evidence for the direct transfer of fructose from sucrose to high M_r glucofructosans was obtained.     

Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography

NP_Δc375 is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self‐assembles into a long helical structure. A packed bed anion exchange chromatography (PB‐AEC), SepFastTM Supor Q pre‐packed column, was used to purify NP_Δc375 from clarified feedstock. This PB‐AEC column adsorbed 76.2% of NP_Δc375 from the clarified feedstock. About 67.5% of the adsorbed NP_Δc375 was successfully eluted from the column by applying 50 mM Tris‐HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB‐AEC operation. Electron microscopic analysis revealed that the helical structure of the NP_Δc375 purified by SepFast^TM Supor Q pre‐packed column was as long as 490 nm and 22–24 nm in diameter. The antigenicity of the purified NP_Δc375 was confirmed by enzyme‐linked immunosorbent assay. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 564–567, 2013     

Affinity chromatography of lactic acid dehydrogenase on N-(6-aminohexyl)oxamate-sepharose

A competitive inhibitor (K_i = 10⁻⁴–10⁻⁵m) for lactic acid dehydrogenase (LDH), N-(6-aminohexyl)oxamate, was synthesized from diethyl oxalate and 1,6-diaminohexane. The affinity column prepared by attachment of this compound to CNBr-activated Sepharose 4B enabled efficient purification of LDH from the marine teleost, Fundulus heteroclitus (Lin.). A similar affinity absorbent prepared by the method of O'Carra and Barry (FEBS Lett. (1972) 21, 281) gave a less satisfactory purification because the LDH was contaminated with phosphorylase a. The reason for this contamination was traced to an incomplete acylation of amino groups on the modified Sepharose beads by the carbodiimide-mediated reaction. Immobilization of the presynthesized affinant, N-(6-aminohexyl)oxamate, assured complete absence of these interfering groups. An alternate synthetic scheme, involving an active ester of ethyl oxalate, was also found to eliminate this problem.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

Spent 99Mo/99mTc generator as an economical source of 99Mo

An improved method for utilization and purification of ⁹⁹Mo from spent ⁹⁹Mo/^99mTc generators has been described. After washing the generator with saline to remove the generated ^99mTc, followed by 2 mL 5 M NaOH containing a few drops of H₂O₂, the ⁹⁹Mo was quantitatively eluted from the generator with 5 mL 5 M NaOH. The alkaline eluate containing ⁹⁹Mo was contaminated with partially dissolved alumina. In the present method, an anion-exchange resin Dowex 1 × 8 column was used for purification of ⁹⁹Mo from the contaminating alumina. The resultant ⁹⁹Mo was of high purity and contained < 1 μg Al³⁺/mL ⁹⁹Mo solution, as estimated by atomic absorption. The eluted ⁹⁹Mo was used for radiolabeling of monoclonal antibodies (Mab) to verify the theoretical modeling of an in vivo generator for radioimmunotherapy, and the results of this study will be published elsewhere.     

Production, Partial Purification and Characterization of Extracelluar Xylanase from Chaetomium globosum

Culture conditions for efficient production of extracellular xylanase by fungus, Chaetomium globosum isolate Cg2, have been standardized. Further, xylanase has been partially purified and characterized. Xylanase activity was maximum after 9 days of incubation when amended in medium with 1.5 % xylan as carbon source and 0.6% NH₄H₂PO₄ as nitrogen source. Partial purification of the xylanase was accomplished by ammonium sulphate precipitation, followed by further purification by anion exchange chromatography on DEAE-Sephadex A-50 column. The partially purified enzyme was electrophoresed on SDS-PAGE and a single band produced corresponded to molecular weight, 32 kD. The optimum temperature and pH for maximum activity of purified xylanase were 30°C and 5.5, respectively. Both the purified xylanase and culture filtrate have shown the antifungal activity against Bipolaris sorokiniana, a causal organism of spot blotch of wheat. Purified xylanase at 100 μg ml^?1 concentration caused 100 per cent inhibition of conidia germination of B. sorokiniana, whereas the culture filtrate was able to inhibit germination up to 67.5 per cent.     

Analysis of the free nucleotide pools of mammalian tissues by high-pressure liquid chromatography

Perchloric acid extracts of tissues were neutralized with tri-N-octylamine and, after removal of ClO₄^?, subjected to preliminary purification on a Cu²⁺-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure was developed and gave good resolution of the naturally occurring free nucleotides on a single column. Where heterogeneous peaks eluted, an effective supplementary analysis was achieved by reverse-phase HPLC. An HPLC paired-ion technique was also evaluated for use in nucleotide analysis. Although anion-exchange was best for overall separation of nucleotides, both reverse-phase and paired-ion chromatography gave excellent separation of cyclic nucleotides. Reduced pyridine nucleotides were detected and measured in the form of their acid-decomposition products. The recovery of nucleotides was examined throughout the described analytical techniques and shown to be quantitative.     

De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons

Germination of peanut seed is accompanied by a rapid increase in isocitritase (isocitrate lyase, EC 4.1.3.1) during the first 4 days. The presence of cycloheximide (50 μg/ml) during water imbibition inhibited the increase in isocitritase activity. Actinomycin D conversely did not inhibit isocitritase activity until the second day of imbibition while RNA synthesis was inhibited. Germination of peanut seed in ¹⁴C-reconstituted amino acids followed by fractionation of a 20 to 35% ammonium sulfate preparation on a Sephadex G-200 column (57-fold purification) showed that the active enzymic fraction coincided with a large peak of radioactivity. Germination of peanut seed in 45% D₂O followed by enzyme purification and CsCl equilibrium centrifugation revealed that all the enzyme from D₂O seed had a higher density than normal isocitritase. These data indicate that isocitritase in peanut seed is synthesized de novo.     

Comparative physico-kinetic properties of a homogenous purified β-glucosidase from Withania somnifera leaf

β-glucosidase from Withania somnifera (Solanaceae) leaf has been purified to homogeneity and characterized for its physico-kinetic properties. The enzyme purification was achieved through a sequence of gel filtration and ion-exchange column chromatography, and PAGE revealed the homogeneity purification status of the enzyme. The properties of the enzyme included an acidic pH optima (4.8), alkaline pI (8.7), meso-thermostabity, monomeric structure with subunit molecular weight of about 50 kDa, high affinity for substrate (K _m) for pNPG (0.19 mM) and high (105,263 M^?1 s^?1) catalytic efficiency (K _cat/K _m). The mesostable enzyme had a stringent substrate specificity restricted only to β-linked gluco-conjugate. The enzyme is optimally active at 40 °C with 12.4 kcal Mol^?1 activation energy, and was highly sensitive to d-gluconic acid lactone inhibition (94 % at 1 mM) with an apparent K _i 0.21 mM. The enzyme could catalyze transglucosylation of geraniol with pNPG as glucosyl donor, but not with cellobiose. Some of the physico-kinetic properties were noted to be novel when comprehensively compared with its counterparts from plant, animal and microbial counterparts. Nevertheless, the catalytic and other features of the enzyme were relatively closer to Oryza sativa among plants and Talaromyces thermophillus among fungi. Significance of building-up of a library of novel plant β-glucosidases for structural investigation to understand naturally evolved mechanistics of catalysis has been indicated.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

A simple procedure for regeneration of an organomercurial agarose column

Organomercurial agarose has been used in the purification of various thiol compounds including enzymes (1). Thiol compounds are first adsorbed on a column of organomercurial agarose, and then eluted with a second thiol compound, e.g., 2-mercaptoethanol (2-ME)¹ and cysteine. Although this column can be used repeatedly, a usual method for regeneration of the column is to remove the second thiol by HgCl₂. It would be desirable to regenerate the column without using HgCl₂, since it is biohazardous. In the study of the purification of a thiol-containing enzyme, we found that organomercurial agarose, which had previously been treated with 2-ME, could adsorb the enzyme and that the enzyme was eluted with 2-ME. This finding led us to examine whether the column can be used repeatedly without the regeneration using HgCl₂.     

Affinity chromatography of Pseudomonas salicylate hydroxylase

In order to facilitate the purification of salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) from Pseudomonas sp. RPP (ATCC 29351), an affinity chromatography procedure was developed employing immobilized salicylate as the affinity ligand. The immobilization was achieved by reacting p-aminosalicylate with the N-hydroxysuccinimide ester of Sepharose 4B-6-aminohexanoic acid. When the bacterial crude extract was chromatographed with this affinity column, salicylate hydroxylase was absorbed to the gel while the bulk of protein freely passed through. The absorbed enzyme was subsequently eluted from the affinity column by applying a 0–60 mm sodium salicylate gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzymatically most active fraction of the affinity effluent revealed salicylate hydroxylase was by far the most predominant protein but there were also small amounts of contaminating proteins. However, a virtually homogeneous enzyme preparation was obtained when the crude extract was first fractionated with a DE-52 anion-exchange column followed by the affinity step. The enzyme preparation obtained by this two-step procedure showed a specific activity of 14.9 units/mg and an A₄₅₀:A₃₇₂:A₂₈₀ of 1.01:1:10.23. Because most of the enzymes belonging to the class of external flavoprotein monooxygenase utilize salicylate analogs as substrates and share many other common properties, there is a strong possibility that the salicylate column may be useful for the purification of other member monooxygenases.     

Initiation of ovalbumin synthesis in chicken oviduct magnum: Transient labeling of the NH2-terminus by methionine

The incorporation of [³⁵S]methionine into ovalbumin, a protein containing NH₂-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [³⁵S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P₀, P₁, and P₂-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P₀ and P₁ showed that the ratio of radioactivity in NH₂-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH₂-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH₂-terminal N-acetylglycine.     

Bacteriophage lambda FII gene protein: role in head assembly

The in vitro completion of bacteriophage lambda F_II^? heads to form phage can be used as an assay for the λ F_II gene protein. F_II protein activity is released from highly purified phage particles or phage heads by treatment with heat or denaturing agents. F_II protein was purified from isolated phage particles and from an extract of E^? infected cells in which it is not bound to any large structures. No differences in molecular weight (11,500), isoelectric point (4.75), electrophoretic mobility, or purification properties could be demonstrated between the F_II proteins from the two sources. Thus the polypeptide does not seem to be modified during assembly.Phage φ80 is closely related to λ. φ80 heads will join to φ80 tails in vitro but will not join to λ tails, though λ heads will join to either type of tail. Mixing experiments between F_II^? heads, tails, and F_II protein from λ or φ80 show that the specificity of head-tail joining is correlated with the source of the F_II protein and not with the source of the other head proteins. Thus, F_II protein is apparently responsible for this specificity of head-tail joining.     

Purification and characterization of beef liver dihydrofolate reductase.

Beef liver dihydrofolate reductase has been purified to homogeneity by using a methotrexate affinity column followed by gel filtration to remove several higher molecular weight proteins. Tightly bound dihydrofolate is removed by hydroxylapatite chromatography. The overall purification is 13,000-fold; the specific activity is 26 units·mg^?1, approximately 25 times higher than previously reported. The enzyme has been shown to be homogeneous by the following criteria: (i) discontinuous gel electrophoresis, (ii) sodium dodecyl sulfate-gel electrophoresis, (iii) velocity sedimentation, (iv) equilibrium sedimentation, and (v) methotrexate titration. The amino acid composition has been determined. Notable features include a single cysteine, three tryptophan and three histidine residues. The N-terminal amino acid is leucine. The molecular weight determined by equilibrium sedimentation is 22,500. The s_20,w⁰ is 2.08 × 10^?13 S and D_20,w⁰ = 10.93 cm²·s^?1. A frictional coefficient of 1.04 indicates that the enzyme is essentially spherical. An isoelectrical point of 6.80 was measured.