Hydroxylapatite thermal fractionation of chromatin and DNA

Chromatin and DNA from developing muscle cultures were fractionated by hydroxylapatite thermal chromatography on the basis of differential thermal stability. A thermal chromatography system was developed in which protein mediated thermal stability of chromatin DNA was maximally expressed. The resulting chromatin and DNA elution profiles were similar to thermal denaturation profiles in low ionic strength solution. Additional studies showed this system was able to detect protein stabilization of DNA in in vitro nucleohistone preparations. Although some protein remained bound to hydroxylapatite during chromatin thermal elution, it did not affect the denaturation or elution behavior of free DNA on the same column. These studies show that fragments of chromatin or DNA can be segregated on the basis of differential thermal stability by hydroxylapatite chromatography.     

Cytological localization of inverted repeated DNA sequences in Vicia faba

Inverted repeated DNA sequences have been isolated from sheared Vicia faba DNA by hydroxylapatite column chromatography, treated with nuclease S₁, tritiated by the nick translation method and hybridized in situ on squashes of Vicia faba root tips. Silver grains appear grouped in a rather limited portion of interphase nuclei and form a sort of band across them. The central regions of metaphase chromosomes are preferentially labeled, labeling being excluded from telomeres, centromeres and secondary constrictions. These results are briefly discussed in relation to those obtained in other species and the functional significance of inverted repeats.     

High-flow-rate hydroxylapatites

We have established a novel and simple method for preparing rigidly crystallized or granulated hydroxylapatites, which showed flow rates one order of magnitude higher in column chromatography than those of the Tiselius specimen (1). The essential difference in the procedures from the Tiselius method is that calcium phosphate, the precursor of hydroxylapatite, is generated not at room temperature but at either 45 or 95 degrees C. The capacities of the two hydroxylapatites for binding and fractionating protein, RNA, and DNA upon column chromatography were similar to those of Tiselius's hydroxylapatite. The 95 degrees C specimen can be dried with no change in the properties. Because of the reproducibly observed properties, including the extremely high flow rate with ease in handling as well as low cost and simple procedures with no additives and with no special equipment in preparation, our hydroxylapatites seem to be best so far for routine laboratory use.     

Isolation of foldback DNA utilizing nuclease S1 digestion in aqueous dioxane.

An improved method for the isolation of the inverted repetitive (foldback) sequences present in mammalian DNAs is described. It makes use of the new observation that nuclease S1 digestion of denatured DNA occurred at a faster rate and was more extensive in medium containing dioxane. The temperature-absorbance characteristics of nuclease S1-resistant DNA were systematically studied as a function of the temperature employed during the step of enzymic hydrolysis. Specimens of human placental and calf thymus DNA which had been denatured and renatured to C₀t ≤ 10^?3 mol s liter^?1 were used as substrates. Foldback DNA was isolated from the enzymic digests by means of hydroxylapatite chromatography. Temperature-absorbance studies showed the enzyme-resistant DNA had a high degree of thermal stability; the hyperchromic rises equaled those obtained in the native speciments. The amount of foldback DNA which could be obtained was not influenced by the fragment size of the starting material, above a certain molecular weight range. Foldback DNA represented about 4% of the human genome and at least 5% of the bovine genome. The size distributions of these strands were studied by means of polyacrylamide gel electrophoresis.     

Sensitive detection of 5-methylcytosine and quantitation of the 5-methylcytosine/cytosine ratio in DNA by gas chromatography--mass spectrometry using multiple specific ion monitoring.

A method combining gas chromatography and mass spectrometry (GC-MS) with multiple specific ion monitoring has been developed for the detection of 5-methylcytosine and the quantitation of the ratio of methyleytosine to cytosine in DNA. The trimethylsilyl derivatives of cytosine and 5-methylcytosine obtained from DNA hydrolysates are separated by isothermal elution on an OV-225 column and detected by specific ion monitoring in a DuPont 321 mass spectrometer. As little as 1.6 pmol of 5-methylcytosine in Φ_χ174 DNA can be detected, corresponding to a tenfold improvement in sensitivity over that obtained by conventional techniques. The ratio of 5-methylcytosine to cytosine of DNA from φ_χ174, calf thymus, salmon sperm, and several mouse tissues has also been determined. The results agree well with those obtained by other methods.     

Purification, properties, and kinetic observations on the isoenzymes of NADP isocitrate dehydrogenase of maize.

A successful method for the purification of NADP isocitrate dehydrogenase from a plant source, Zea mays, is reported. Two mitochondrial isoenzymes were found and purified to homogeneity by a course of acetone fractionation, bulk exchange on DEAE-cellulose, cellulose hydroxylapatite column chromatography, and continuous elution electrophoresis. The mitochondrial isoenzymes are very similar with respect to kinetic properties, response to solvent perturbation, and temperature dependence of the pH/V relationship of isocitrate dehydrogenation. The Michaelis constant for isocitrate is identical for both isoenzymes. The enzymes have a molecular weight of 81,000 as estimated by permeation chromatography and an isoelectric point of 5.5 as extrapolated from gel-electrophoretic mobilities. Detectable differences are confined to differences in electrophoretic mobilities and heat denaturation. In D₂O the rate of the overall reaction from isocitrate to α-ketoglutarate and CO₂ was about 3.6 times slower than the same reaction in H₂O. Both the forward and reverse reactions, in which isocitrate is dehydrogenated or generated from oxalosuccinate, were observed to decrease by this amount in D₂O. The decarboxylation of oxalosuccinate was found to decrease by only about 25% in D₂O relative to the velocity of the reaction in H₂O. Thus the slow step in the overall reaction must be the initial dehydrogenation step rather than the decarboxylation of oxalosuccinate. The pK of the overall reaction did not change in D₂O as compared to H₂O.     

Seasonal change and prolonged anoxia affect the kinetic properties of phosphofructokinase and pyruvate kinase in oysters

The effects of seasonal change, November versus July, and prolonged anoxia (96 h under N₂ gas) on the properties of phosphofructokinase and pyruvate kinase from five tissues (gill, mantle, hepatopancreas, phasic adductor, catch adductor) of the oyster, Crassostrea virginica, were investigated. Both enzymes showed tissue-specific and season-specific changes in kinetic properties; for pyruvate kinase this correlated with seasonal differences in enzyme elution patterns on hydroxylapatite chromatography. Kinetic properties of both enzymes in winter were consistent with primarily catabolic roles in glycolysis with responsiveness to cellular energy demands, whereas in summer these enzymes may be more closely regulated with respect to the biosynthetic and gluconeogenic functions of the tissues. Anoxia-induced changes in phosphofructokinase properties were relatively minor but anoxia stimulated changes in pyruvate kinase properties and elution profiles on hydroxylapatite in all tissues except mantle, with much greater effects seen for the enzyme from winter versus summer animals. For example, anoxia-induced changes in pyruvate kinase from winter gill included a fourfold rise in the substrate affinity constant for phosphoenolpyruvate, a sevenfold increase in the concentration of fructose-1,6-bisphosphate needed to activate the enzyme by 50%, and a 50% decrease in the concentration of L-alanine that inhibits activity by 50%. Changes in pyruvate kinase kinetics and hydroxylapatite elution patterns during prolonged anoxia are consistent with covalent modification of pyruvate kinase but contrary to results for many other mollusc species, anoxia exposure appears to induce a dephosphorylation of the enzyme. Accepted: 17 February 2000     

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound ah-sepha-rose 4b

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono(“phospho”)-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo D-glucose (GlcN-6S), as well as (δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-α-L-thero-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxyla patite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxyl- patite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.     

A Two-Step Procedure for Quantitative Isolation of Pure Double Strand DNA from Animal Tissues and Cell Cultures

A two step procedure for the quantitative isolation of protein- and RNA-free double-strand DNA from animal tissue and cell homogenates is described. In the first step proteins not complexed with DNA are hydrolyzed with an immobilized protease (Proteinase K) that is separated by filtration after the de-proteinization. Then the DNA is adsorbed to hydroxylapatite (HA) and desorbed from the adsorbent by stepwise elution with buffers of increasing ionic strength. The DNA content was determined directly from the absorption at 260 nm. The melting curve of the isolated DNA showed that it was double stranded. The protein content in the DNA was determined from the ratio of the adsorbance at 260 to 230 nm. Non-histone proteins complexed to DNA determined the rate of deproteinization that was found to be tissue specific. These proteins were found to have a larger influence on the ratio A₂₆₀/A₂₃₀ than histones, indicating that their absorption (at 230 nm) is markedly perturbed when they are bound to DNA.     

A hydroxylapatite batch assay for quantitation of cellular DNA damage.

A batch elution procedure is described for quantitative measurement of DNA damage. The technique is based on alkaline unwinding of cellular DNA and separation of singlestranded from duplex forms by step elution from hydroxylapatite with phosphate formamide. The method is rapid, permits large numbers of samples to be handled simultaneously, and consistently yields recoveries of >95% of total chromatographed DNA. Because as many as 1 × 10⁷ cells per batch may be analyzed, quantitation of the eluted DNA by nonradioactive methods is feasible. The method is standardized with respect to the unwinding constant β, the alkaline DNA unwinding unit Mng, and the DNA-damaging efficiency of ionizing irradiation.     

Zonal rate centrifugation of proteoglycans in sucrose gradients

A simple reverse-phase chromatographic system for separating deoxyribonucleoside monophosphates is described. Using isocratic elution at room temperature, clear separation of seven of the deoxyribonucleoside monophosphates that occur in either procaryotic or eucaryotic DNAs can be achieved in less than 2 h. Thus, this method allows a sensitive and rapid analysis of submicrogram quantities of ³²P-labeled deoxyribonucleoside monophosphates derived from DNA labeled in vivo with ³²P_i or from DNA labeled enzymatically in vitro at the 5′ or 3′ ends. The suitability of the method for studying methylation of mammalian DNAs is illustrated by presenting examples of its application to (a) quantitation of major and minor nucleotides in newly synthesized DNA, (b) determination of the specificity of in vitro methylation of DNA, and (c) quantitation of the extent to which specific restriction endonuclease sites are methylated in vivo.     

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.     

Properties of the soluble arachidonic acid 15-lipoxygenase and 15-hydroperoxide isomerase from the oomycete Saprolegnia parasitica

The soluble hydroperoxide isomerase and 15-lipoxygenase activities were partially purified from the oomycete Saprolegnia parasitica and some of their properties characterized. Both enzymes co-eluted with a molecular weight of 145,000–150,000 on Sephacryl S-300 chromatography. The enzyme activities also co-eluted on DEAE Sephadex ion exchange chromatography and hydroxylapatite chromatography. Both activities showed similar responses to pH and temperature. Both enzymes showed parallel inhibition by p-hydroxymercuribenzoate and eicosatetraynoic acid. The partially purified hydroperoxide isomerase showed an apparent k_m of 166 μM and a V_max of 5.3 μmol/min/mg protein for exogenous 15-HPETE. It was not stimulated by calcium. These results suggest that the soluble hydroperoxide isomerase and 15-lipoxygenase activities from S. parasitica are both contained on the same protein or protein complex.     

Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification

ABSTRACT

As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, Z_Ca, displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of Z_Ca to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, Z_CaTetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with Z_CaTetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.     

Single-strand-specific degradation of DNA during isolation of rat liver nuclei

We have investigated structural change in rat liver DNA produced by different isolation procedures and specifically compared the integrity of DNA derived by phenol extraction from isolated and purified nuclei with preparations extracted immediately from a crude liver homogenate containing intact nuclei. As indicated by stepwise elution from benzoylated DEAE-cellulose, most structural change in DNA was evident following nuclei isolation. Damage principally involved generation of single-stranded regions in otherwise double-stranded DNA fragments; totally single-stranded DNA was not detected by hydroxylapatite chromatography. Caffeine gradient elution suggested formation of single-stranded regions extending for up to several kilobases. In neutral sucrose gradients, differences in sedimentation rates of respective DNA samples consequent upon S1 nuclease digestion could be detected after isolation of nuclei, though not in other circumstances. The observed single-strand-specific nuclease digestion of DNA could apparently be reduced if steps were taken to reduce autodigestion during nuclei isolation by reduction of temperature and covalent cation concentration. The results are discussed in terms of the use of exogenous and endogenous nucleases in chromatin fractionation studies involving isolated nuclei and possible artifactual findings that may be generated by single-strand-specific autodigestion.     

Physico-chemical model for DNA alkaline elution: New experimental evidence and differential role of DNA length,chain flexibility and superpacking

For a better understanding of data provided by DNA alkaline elution technique, a new analytical model has been developed which takes into consideration both the physicochemical properties of in situ DNA strand (length and flexibility/superpacking) and the geometric and hydrodynamic configuration of the elution apparatus (flow and filter conditions). Simulation by this model of experimental data previously obtained before and after carcinogens administration, has shown that for constant flow and filter conditions elution profiles are dependent, not only from DNA molecular weight, but also from a parameter critically related to modifications in chain flexibility/superpacking. This has been confirmed by several independent observations, including the time-dependent changes in non-denaturing lysing solution monitored by hydroxylapatite and alkaline elution techniques.     

Further purification of bovine spleen inhibitors of lymphocyte DNA synthesis (chalones)

Partially purified lymphocytic factors were obtained from bovine spleen; these factors are non-cytotoxic and biologically active in vitro and in vivo:[³H]Thymidine incorporation into DNA of mitogen-stimulated mouse spleen cells in culture is inhibited: similar results are obtained with phytohaemagglutinin-stimulated peripheral human lymphocytes, where blast cells transformation is blocked.[³H]Thymidine incorporation into DNA of mitogen stimulated lymphocytes withdrawn from in vivo treated mice, is also reduced.The two factors in vitro and in vivo seem to act preferentially on mouse spleen cells compared to their action on liver, kidney and testicle cells in culture as far as thymidine incorporation into DNA is concerned.The following techniques were applied for their purification: 1. Homogenization of the fresh tissue in water, centrifugation, dialysis of the supernatant, centrifugation fractionation of the supernatant by alcoholic precipitation and finally concentration in vacuo and lyophilization of the material soluble at 75% of alcohol yielded fraction F. 2. Preparation of an acetone powder from the spleen, extraction of the dry powder with water, then high speed centrifugation, followed by lyophilization of the supernatant produced fraction F′.Both fractions F and F′ were further fractioned by chromatography on a Sephadex G75 column: 7 peaks were obtained (F₁–F₇). Biological activity was found in fraction F₁, corresponding to high molecular weight material, and in fraction F₁, corresponding to low molecular weight substances.By rechromatography on Sephadex G 75, it is easy to dissociate from F₁ a small molecular weight fraction which might be similar to F₆ as far as elution volume and biological properties are concerned.     

DNA fragmentation in N-diazoacetylglycine amide-treated cells determined, by the rate of strand separation in alkali with hydroxylapatite chromatography in batch

DNA damage induced in mammalian cells (CHO-K1) by one hour treatment with several concentrations of N-diazoacetylglycine amide (DGA) was evaluated by the method of DNA denaturation in alkali and successive neutralization followed by separation of single from double stranded DNA with the recently described technique of hydroxylapatite chromatography performed in batch. This latter technique does not need complex apparatus and simplifies the simultaneous handling of large number of samples; it also appears as sensitive and reliable as the DNA alkaline elution on filter, to which it can be regarded as both alternative and complementary.     

One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata

Phycoerythrins have been widely used in food, cosmetics, immunodiagnostics and analytical reagents. An efficient one-step chromatography method for purification of R-phycoerythrins from Polysiphonia urceolata was described in this paper. Pure R-phycoerythrin was obtained with an absorbance ratio A(565)/A(280) of 5.6 and a high recovery yield of 67.33% using a DEAE-Sepharose Fast Flow chromatography with a gradient elution of pH, alternative to common gradient elution of ionic strength. The absorption spectrum of R-phycoerythrin was characterized with three absorbance maxima at 565, 539 and 498 nm, respectively and the fluorescence emission spectrum at room temperature was measured to be 580 nm. The results of native-PAGE, and SDS-PAGE showed no contamination by other proteins in the phycoerythrin solution, which suggests an efficient method for the separation and purification of R-phycoerythrins from Polysiphonia urceolata.     

Indolethylamine N-methyltransferases of Phalaris tuberosa, purification and properties

Two S-adenosylmethionine-dependent indolethylamine N-methyltransferase activities were purified from soluble extracts of Phalaris tuberosa by fractionation with (NH₄)₂SO₄, and chromatography on DEAE-Sephadex. One enzyme methylated the primary indolethylamines and the other methylated the secondary indolethylamines in the plant. These two enzymes were similar in catalytic and bulk physical properties and could not be separated during purification or by A 1.5 Agarose or hydroxylapatite chromatography. The products of enzymic reactions were identified by TLC.