Gel Filtration Method Research Articles

Characterization and potential application of purified aldehyde oxidase from Pseudomonas stutzeri IFO12695

The molecular weight of purified aldehyde oxidase from Pseudomonas stutzeri IFO12695 was estimated to be 160 kDa by a gel filtration method. SDS-PAGE showed that the enzyme consisted of three non-identical subunits with molecular weights of 18, 38, and 83 kDa. The enzyme exhibited an absorption spectrum with maxima at 277, 325, 365, 415, 450, 480, and 550 nm and possessed molybdenum, CMP, iron, sulfur, and FAD as its cofactors, indicating that it belonged to the xanthine oxidase family. A variety of aliphatic and aromatic aldehydes were oxidized; and among them n-hexylaldehyde gave the most rapidly action. When 10 mM formaldehyde was treated with the aldehyde oxidase in the presence of catalase for 240 min, the formaldehyde concentration was reduced to 0.8 mM, suggesting this enzyme might be effective for the removal of formaldehyde contained in wastewater.

Micellar Counterion Binding Probed by Fluorescence Quenching of 6-methoxy-N-(3-sulfopropyl)quinolinium

The halide sensitive fluorescence probe, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), provides a direct approach for measurement of halide concentrations dissociated from the surfactant monomer and micelle. The gel filtration method confirms that the water-soluble SPQ probe will tend to partition in aqueous bulk phase without trapping in cationic micelles. The fluorescence of SPQ is quenched by free halide ions with a linear Stern-Volmer relation below the CMC of cationic surfactants. The Stern-Volmer plot gave a distinct break at CMC owing to the counterion binding to micelles. The CMC and degree of micellar counterion binding (β) are generally in agreement with those obtained by conductivity method. The SPQ fluorescence quenching had high sensitivities with Stern-Volmer constants of 125 M-1 (chloride) and 223 M-1 (bromide), while it was insensitive to nitrate ion. The selective quenching behavior shows that the nitrate ions bind to micelles displacing bromide for tetradecyltrimethylammonium micelles. The increase in counterion binding to micellar surface lower the CMC of surfactants, TTAOAc (tetradecyltrimethylammonium acetate) > TTAC (tetradecyltrimethylammonium chloride) > TTAB (tetradecyltrimethylammonium bromide) > TTAN (tetradecyltrimethylammonium nitrate).

Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides.

We present a fast and simple protocol for large-scale preparation and purification of RNA oligonucleotides. RNA oligonucleotides are prepared by in vitro transcription with T7 RNA polymerase from linearized plasmid DNA templates constructed by PCR. In place of denaturing polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography is employed to purify the RNA oligonucleotide from the transcription mixture yielding >99% pure RNA product. In contrast to PAGE-based purification, the gel filtration method does not require denaturation of the RNA oligonucleotide, which is desirable for larger RNAs, and the product is free of low-molecular-weight acrylamide contaminants, which greatly benefits NMR, crystallographic, and other biophysical studies of large RNAs and RNA-protein complexes.

The periplasmic E. coli chaperone Skp is a trimer in solution: biophysical and preliminary crystallographic characterization.

The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria. Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins. Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution. Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions. Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found. A first data set was collected to 2.60 A resolution.

Tetrapandins, a New Class of Scorpion Toxins That Specifically Inhibit Store-operated Calcium Entry in Human Embryonic Kidney-293 Cells

Venoms from 14 snakes and four scorpions were screened for inhibitory activities toward store-operated Ca2+ entry (SOCE) in human embryonic kidney-293 cells. An inhibitory activity was found in venom from the African scorpion Pandinus imperator. The active agent of this venom was purified by gel filtration and reverse-phase high pressure liquid chromatography methods. Sequence information on the purified fraction, by automatic Edman degradation and mass spectrometry analysis, identified the activity as being contained in two tetrapeptides, which we have named tetrapandins. We demonstrate that synthesized tetrapandins have inhibitory activity for SOCE in human embryonic kidney-293 cells while having no effect on either thapsigargin- or carbachol-stimulated release of Ca2+ stores. These toxins should be extremely useful in future studies to determine downstream events regulated by SOCE as well as to determine whether multiple pathways exist for thapsigargin-stimulated Ca2+ entry.

A simple cost-effective screening test for microalbuminuria

Estimation of urinary albumin excretion in micro quantities requires expensive immunochemical methods which are not freely available in developing countries with limited resources. A study was performed to set up a simple cost-effective test to quantitatively assess the microalbuminuria. Urine specimens (173 samples) were collected and albumin concentration was estimated by precipitating with 25% sulphosalicylic acid (SSA) and use of a light dependent resistor (LDR) system. As the sensitivity of LDR system was interfered by the colour pigment of urine, all samples were run through a Sephadex G-25 micro column to separate the colour pigment. The present test detects albumin concentrations in the range 10-200 mg/L with a sensitivity and specificity values of 83 and 79% respectively. We conclude that the present LDR micro column gel filtration method can be used as a cost -effective screening test for microalbuminuria in developing countries.

Separation and characterization of metallothionein in the liver of sea turtles by high performance liquid chromatographylinductively coupled plasma-mass spectrometry

To investigate whether trace metals bind to metallothioneins (MTs) in the hepatocytosol of green turtles (Chelonia mydas) and hawksbill turtles (Eretmochelys imbricata), MT fraction was obtained by ultracentrifugation and gel filtration methods. MTs separated from hepatocytosol were further purified and characterized by high performance liquid chromatography/inductively coupled plasma-mass spectrometry. In addition, the involvement of MTs in the accumulation of trace metals in the liver of sea turtle was examined. Gel filtration analysis showed that significant amounts of Cu, Zn, Ag and Cd were bound to MT in the cytosol of sea turtles, suggesting that such trace metals were primarily detoxified by interaction with MTs in the liver. Elution profiles of these trace metals by anion-exchange chromatography were different between green turtles and hawksbill turtles. These results suggest the presence of multiple isoforms of MT in the liver of both sea turtles; however, constituents of isoforms were different between green and hawksbill turtles. In both species, we observed the elevation of the height of a specific peak in elution profile with an increase in Cu concentration in hepatocytosol. This result suggests the presence of a novel MT isoform related to copper accumulation in the liver of sea turtles.

Inhibition of Hemostasis by a High Affinity Biogenic Amine-binding Protein from the Saliva of a Blood-feeding Insect

The saliva of the blood-feeding insect Rhodnius prolixus contains numerous pharmacologically active substances. Included among these are a number of lipocalin proteins that bind various ligands important in hemostasis and inflammation. One such protein is a biogenic amine-binding protein (ABP) that binds serotonin, epinephrine, and norepinephrine. Based on amino acid alignments, it is most similar to the nitrophorin group of lipocalins found in the same insect species. Physiologically, this protein appears to act as both a vasodilator and platelet aggregation inhibitor. This protein inhibits smooth muscle contraction of the rat uterus in response to serotonin and of the rabbit aorta in response to norepinephrine. Platelet aggregation induced by a combination of low concentrations of ADP and either serotonin or epinephrine is inhibited because of the binding of serotonin and epinephrine. Potentiation of aggregation induced by low concentrations of collagen along with serotonin or epinephrine is also inhibited. Dissociation constants for biogenic amines were measured using isothermal titration calorimetry and the Hummel-Dreyer method of equilibrium gel filtration. In this manner, K(d) values of 102, 24, and 345 nm were found for serotonin, norepinephrine, and epinephrine, respectively. Molecular modeling of ABP suggests that ligand binding is mediated by interaction with the side chains of aromatic amino acids and charged residues that line the binding pocket.

Microplate gel-filtration method for radioligand-binding assays

A thin-layer gel-filtration chromatographic method has been developed in a 96-well format to separate free and protein-bound ligand in radioligand-binding assays. The mobile phase in the gel-filtration plate is removed via quick centrifugation before samples are applied. Protein-bound ligand is recovered via centrifugation into another 96-well plate for radioactivity measurements. The method exhibits excellent recovery of protein-ligand complexes and less opportunity for dissociation of the complexes since it eliminates major dilution effects from the mobile phase of a column and from elution steps in conventional gel-filtration chromatography. It offers other advantages: simple, rapid, inexpensive, quantitative, and able to handle a large number of samples as required in drug discovery and clinical settings. This microplate gel-filtration method was optimized in studies of receptor-ligand interactions using estrogen receptors as examples and can be used in other radioligand-binding assays.

Free cortisol index as a surrogate marker for serum free cortisol.

The biologically active component of a hormone is the unbound or free fraction. Changes in cortisol-binding protein could give misleading results if only total cortisol is measured for the interpretation of dynamic function tests. This study aimed to measure serum free cortisol using a steady-state gel-filtration method and then to evaluate the correlation between the serum free cortisol and the free cortisol index (FCI), defined as serum total cortisol/cortisol-binding globulin (CBG). Forty-eight serum samples from healthy volunteers undergoing a short Synacthen test were analysed for total cortisol, free cortisol and CBG. The FCI correlated well with a previously established, but more complex, calculation of serum free cortisol (R = 0.98, P <0.001) and with measured serum free cortisol (R = 0.90, P < 0.001). Free cortisol index is a reliable and user-friendly measure of serum free cortisol.

Biochemical and functional characterization of recombinant Rhodnius prolixus platelet aggregation inhibitor 1 as a novel lipocalin with high affinity for adenosine diphosphate and other adenine nucleotides.

The Rhodnius prolixus aggregation inhibitor 1 (RPAI-1) is a novel blood-sucking salivary molecule that binds to ADP and attenuates platelet aggregation. In this report, we determine the binding constants and specificity of RPAI-1 for adenine nucleotides and its functional significance. By the Hummel-Dreyer method of equilibrium gel filtration, we show that RPAI-1 binds ADP with a K(0.5) of 48.6 plus minus 12.2 nM. RPAI-1 also displays high-affinity binding to ATP, AMP, Ado, AP4A, and alpha,beta Met ADP; however, RPAI-1 does not bind to inosine, guanosine, uridine, or cytidine. Binding is not modified by EDTA, indicating that Ado structure but not phosphate groups or Ca(2+) is necessary for binding. By computer simulation, we show that RPAI-1 is more effective in scavenging low but not high concentrations of ADP, in contrast to R. prolixus apyrase. RPAI-1 inhibits in vitro the ADP-dependent platelet-rich plasma aggregation by collagen (COLL), TRAP, PAF, and A23187 but did not block platelet aggregation by ristocetin or phorbol myristate acetate (PMA) and only slightly attenuated that by convulxin. RPAI-1 prolongs the closure time as assessed with PFA-100, when COLL-Epi but not COLL-ADP cartridges are employed. RPAI-1 also affects platelet-mediated hemostasis time and COLL-induced thrombus formation at high shear as assessed with the Clot Signature Analyzer. We conclude that RPAI-1 exerts an antiplatelet effect due to scavenging of low concentrations of ADP in vitro and in vivo. RPAI-1 is the first lipocalin described so far with unique specificity for adenine nucleotides.

DNA Polymerase-Associated and Autonomous 3"→5" Exonucleases in Invertebrates, Unicellulars, and Bacteria

Recently we have revealed a high content of autonomous 3"→5" exonucleases (AE), i.e., those not bound covalently with DNA polymerases, in cells of vertebrates, from fish to human [1]. In the present work, using gel filtration method, cell-free extracts were studied from 15 objects located at different positions on the phylogenetic tree, such as archaebacteria, eubacteria, fungi, infusorians, coelenterates, annelids, and arthropods. It is shown that enzymatic activity of AE accounts for from 30 to 88% of the total 3"→5" exonuclease activity of the extracts. A part of AE is revealed in zone of high-molecular DNA polymerases and can be separated by change of the chromatography conditions. It indicates a probable formation of complexes of AE with DNA polymerases. The high AE activity in cells of different organisms, from archae- and eubacteria to human, allows suggesting these enzymes to play a significant role in correction of polymerase errors in the processes of DNA replication and reparation, as well as in postreplicative correction of heteroduplexes in DNA.

In vitro digestibility of globulins from cowpea ( Vigna unguiculata) and xerophitic algaroba ( Prosopis juliflora) seeds by mammalian digestive proteinases: a comparative study

Globulins were purified from mature and immature cowpea and algaroba seeds by gel filtration (S-200), anion exchange (DEAE-Sepharose) and other chromatographic methods. These globulins (native and heated) were submitted to hydrolysis by pepsin, chymotrypsin and trypsin and their digestion products were analysed by SDS-PAGE. Results showed that native globulins of both legumes were weakly digested by pepsin and were not digested by serine proteases. Heated proteins from cowpea were digested more rapidly by pepsin and chymotrypsin than was algaroba globulin. Trypsin rapidly digested proteins from both cowpea and algaroba. Comparing the hydrolysis of bean globulins to the algaroba globulins by mammalian enzymes, the globulins from immature cowpea showed better digestibility than mature cowpea; globulins from algaroba pod, which is used as an alternative food, were difficult to digest.

Human Serum Albumin Binding of Novel Antiretroviral Nucleoside Derivatives of AZT

The binding of novel nucleoside derivatives (2–7) to the Human Serum Albumin (HSA) was studied using zidovudine (AZT), as standard compound. The applicability of two different techniques to separate unbound drug from drug–protein complex was analyzed: the gel filtration and ultrafiltration methods. Ultrafiltration was found to be an adequate procedure for the separation of unbounded drug from the drug-protein complex. Incubation temperature ranging from 0 to 37°C did not modify considerably the bound fractions. The same effects were observed as HSA concentration was modified. Binding assays of studied compounds to purified 1% (w/v) HSA at 0°C, indicate that bound fraction of 2–7 ranges from 13 to 47%, exhibiting a higher affinity to HSA than AZT (12%), which would introduce some interesting improvements in their pharmacokinetic properties. In addition, by means of displacement studies using HSA site specific drugs such as diazepam and salicylate, it was determined that AZT binds to site I of the HSA molecule, by a mainly entropy driven process (ΔS = 10.834 cal/mol °K), being these observations extensive to 2–7. Some structural basis to explain enhanced affinity of these novel derivatives was also established.

Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes.

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively.

Analysis of total inositol phosphates in municipal solid waste compost-treated soils by two extraction methods

Humic substances are the most dynamic component of agricultural soils. In this study, humic substances were extracted from soils based on standard acid/base solubility and by gel filtration using Sephadex G25. Organic P is a component of humic substances, and inositol phosphates are considered to have high prominence in organic P. The objectives of the study were to determine the effects of municipal solid waste (MSW) compost on the amount of inositol phosphates (IP) present in soils, and compare methods of extracting humic substances from soils. Total IP (IPT) in soil extracts was determined following separation using a AG-1× 8 anion exchange resin, acid digestion and ICAP analysis for P. The percentage of IP (%IP) of the total P in the soil was also determined by colorimetric analysis. The soils were part of an experiment to study the effects of three rates of MSW compost and fertilizers on three crops grown in a Pugwash sandy loam (Humo-Ferric Podzol). The data were compared using ANOVA atP≤0.05; extraction methods, fractions [humic/fulvic, high/low molecular (MW)], and five treatments (three different rates of compost, fertilizer, and untreated soil) were compared. The gel filtration method extracted significantly more IPT and %IP than the solubility method. In the solubility method, humic and fulvic acids contained the same amounts of IPT and %IP, while the high MW fraction always contained more IPT and %IP than the low MW fraction in the gel filtration method. Fulvic acids and the low MW fraction contained similar amounts of IPT and %IP. There were differences in %IP between compost-treated soils and the non-compost-treated soils, although there were no differences in IPT due to rate of compost addition.

Comparison of Platelet Aggregability and P-selectin Surface Expression on Platelets Isolated by Different Methods

Three methods commonly used for isolation of blood platelets from plasma were compared. Platelets were isolated by: 1) a washing method; 2) a method of metrizamide–gradient centrifugation; 3) a modified method of gel-filtration. The last method employed BSA–Sepharose gel instead of routinely used Sepharose gel saturated with BSA. BSA–Sepharose gel was prepared by covalent binding of thermally deactivated BSA to CNBr-activated Sepharose 2B. In contrast to platelets isolated by the other methods, an aggregability of the gel-filtered platelets and control platelets in plasma, both activated with ADP, were comparable. When expression of P-selectin on the surface of freshly isolated platelets was examined, the gel-filtered platelets exhibited the same extent of fluorescence signal as platelets in the citrated blood, whereas platelets isolated by the other methods exhibited twice the extent of the signal. The methods involving the centrifugation process cause a low but a significant platelet activation.

DNA Polymerase-associated and autonomous vertebrate 3′→5′ exonuleases

Using methods of gel filtration and ultracentrifugation, cell-free extracts from 12 objects representing the main vertebrate representatives (bony fish, amphibian, reptiles, birds, mammals, including human) were studied. The enzyme activity of autonomous 3′→5′-exonucleases (AE) has been established to be 25–90% of the total 3′→5′ exonuclease activity of the extracts. A part of the AE is revealed in a zone of the DNA polymerases of the α-family and can be separated by changing Chromatographic conditions or by repeated fractionation. The high activity of AE allows suggesting their substantial participation in the replicative correction of the DNA-poly-merase errors as well as in the postreplicative correction of the heteroduplexes in the vertebrate DNA.

Hydrophobic interactions of phenoxazine modulators with bovine serum albumin

The interaction of 10-(3′-N-morpholinopropyl)phenoxazine [MPP], 10-(4′-N-morpholinobutyl)phenoxazine [MBP], 10-(3′-N-morpholinopropyl)-2-chlorophenoxazine [MPCP], 10-(3′-N-piperidinopropyl)-2-chlorophenoxazine [PPCP] or 10-(3′-N-morpholinopropyl)-2-trifluoromethylphenoxazine [MPTP] with bovine serum albumin (BSA) has been studied by gel filtration and equilibrium dialysis methods. The binding of these modulators, based on dialysis experiments, has been characterized using the following parameters: percentage of bound drug (Β), the association constant (K 1), the apparent binding constant (k) and the free energy change (δF‡). The binding of phenoxazine derivatives to serum transporter protein, BSA, is correlated with their octanol-water partition coefficient, log10 P. In addition, effect of the displacing activities of hydroxyzine and acetylsalicylic acid on the binding of phenoxazine derivatives to albumin has been studied. Results of the displacement experiments show that phenoxazine benzene rings and tertiary amines attached to the side chain of the phenoxazine moiety are bound to a hydrophobic area on the albumin molecule.

Interaction of substituted phenoxazine chemosensitizers with bovine serum albumin

The binding of 10-[3′-[ N-bis(hydroxyethyl)amino]propyl]phenoxazine [BPP], 10-[3′-[ N-bis(hydroxyethyl)amino]propyl]-2-chlorophenoxazine [BPCP], 10-[3′-[ N-bis-(hydroxyethyl)amino]propyl]-2-trifluoromethylphenoxazine [BPFP], 10-(3′- N-pyrrolidino propyl)-2-chlorophenoxazine [PPCP] or 10-(3′- N-pyrrolidinopropyl)-2-trifluoromethyl-phenoxazine [PPFP] to bovine serum albumin (BSA) has been measured by gel filtration and equilibrium dialysis methods. The binding of these modulators to bovine serum albumin based on dialysis experiments has been characterized by the following parameters: percentage ( β) of bound drug, the association constant ‘ K 1’, the apparent binding constant ‘ k’ and the free energy Δ F°. The binding of phenoxazine derivatives to bovine serum albumin is correlated with their octanol–water partition coefficient, log 10 P. In addition, the displacing activity of hydroxyzine and acetylsalicylic acid on the binding of phenoxazines to albumin has been studied. The results of the displacing experiments showed that the phenoxazine benzene rings and the tertiary amines attached to the side chain of the phenoxazine moiety are bound to a hydrophobic area on the albumin molecule.