Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu²⁺-IMAC column with an increasing gradient of a counter ion (NH⁺₄) run in combination with an increasing pH gradient (6.8–7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.     

Heterogeneity of the calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

1. The Ca²⁺-dependent phosphatidylinositol phosphodiesterase (phospholipase C-type) from the cytosolic supernatant of rat brain was active against exogenous [³²P]-phosphatidylinositol from pH5.0 to pH8.5. However, the activity in the range pH7.0–8.5 could not be recovered after precipitation with (NH₄)₂SO₄; most of the enzyme activity was recovered in the 30–50% fraction and showed a single sharp pH optimum at 5.5. 2. The cytosolic supernatant was analysed by isoelectric focusing on acrylamide gels, and assay at pH5.5. Four peaks of phosphodiesterase activity were found at pI ranges 7.4–7.2, 6.0–5.8, 4.8–4.4 and 4.2–3.8. 3. The cytosolic supernatant was also applied to a chromatofocusing column, and again assayed at pH5.5. Four peaks were eluted: minor, but consistent, activity at the beginning of the elution with a pI of near 7.2 or above; a second peak at pH6.0–5.85; a third broad peak with a wide range pH5.3–4.2; and a fourth peak, which was eluted by washing the column with 1m-NaCl, suggesting an isoenzyme with a pI below 4.0 (supported by the result of the isoelectric focusing). 4. If all the chromatofocusing fractions were assayed at pH7.0 or 8.0 (at 1mm-Ca²⁺), only a single sharp peak was detected, with a pI of 4.6–4.8. This peak disappeared on (NH₄)₂SO₄ fractionation (30–50%) of the cytosolic supernatant, whereas the four peaks with activity at pH5.5 were virtually unaffected. 5. The four activities (assayed at pH5.5) separated by chromatofocusing produced inositol 1:2-cyclic monophosphate, inositol 1-monophosphate and diacylglycerol as enzymic products. 6. We conclude that the Ca²⁺-dependent phosphatidylinositol phosphodiesterase exhibits considerable heterogeneity, both with respect to pH optima of activity, and its isoelectric properties.     

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.     

Determination of a novel indolylpiperazine anti-migraine agent in rat, monkey, mouse and rabbit plasma by high-performance liquid chromatography with electrochemical detection

A specific, accurate, precise and reproducible assay for the quantitation of a novel indolylpiperazine anti-migraine agent (I) in plasma from various animal species is described. The method involves addition of internal standard (I.S.) and 1.0 M sodium carbonate to the plasma sample, vortex-mixing and extraction with ethylene dichloride. The organic layer is then back-extracted in a buffer consisting of 0.1 M tetramethylammonium hydroxide (TMAH), pH 3.0 and 0.1 M (NH₄)₂HPO₄, pH 3.0, in water. The aqueous layer is injected on to a Zorbax cyano analytical column with a mobile phase consisting of acetonitrile, methanol and water (15:5:80, v/v/v) with 0.01 M TMAH, pH 3.0 and 0.01 M (NH₄)₂HPO₄, pH 3.0. The eluate is monitored by electrochemical detection at 0.9 V (guard cell), 0.5 V (detector 1) and 0.8 V (detector 2). The retention times of I and I.S. were 7 and 10 min, respectively. In drug-free control plasma, there were no interfering peaks seen at the retention times of I or I.S. The standard curve was linear over the concentration range of 5–500 ng/ml in rat, monkey, mouse and rabbit plasma. The lower limit of quantitation in all four matrices was 5.0 ng/ml. Within- and between-assay variability of quality control samples was less than 9% relative standard deviation and the predicted concentration of the quality control samples deviated by less than 15% from the nominal concentration. The stability of I was established for up to 36 h in the autosampler tray, up to 10 months in plasma at −20°C and up to 2 h in plasma at room temperature. The assay is validated for determination of I in plasma.     

Crystallization of coupling factor 1 (CF1) from spinach chloroplast.

Spinach chloroplast coupling factor (CF₁) was crystal-lized at 20°C from 0.05 M TRIS-PO₄, containing 4 mM ATP, 15mM KCl, 1.0 mM EDTA and 1.80 M (NH₄)₂SO₄, at pH 7.8. Some unit cell parameters were determined by electron microscopy and by X-ray diffraction. The cube shaped crystals have a tetragonal lattice, a = b = 135 Å, c = 280 Å with eight molecules per unit cell; possible space group P422 or P4₂2₁2, hence half a molecule in the asymmetric unit. Crystals grown at pH 7.5 in the absence of ATP have an orthorhombic lattice, a = 125 Å, b = 145 Å, c = 169 Å (C222₁), eight molecules per unit cell.     

Extraction and quantification of nicardipine in human plasma

A novel simple method of extraction, separation, identification and quantification of nicardipine in human plasma samples was completely studied. The human plasma samples were initially purified by solid-phase extraction (SPE) using a C₁₈ cartridge. The extracted samples were separated and nicardipine present in the samples was quantified by high-performance liquid chromatography (HPLC) on a reversed-phase C₁₈ column employing a mobile phase consisting of 60% (v/v) acetonitrile in 0.02 M NaH₂PO₄ with pH of 6.3 and a variable wavelength UV detector set at 254 nm. The recovery of nicardipine from plasma samples using selective SPE was 91±6.0% and had less interfering compounds in the HPLC analysis compared to the use of liquid–liquid (L/L) extraction. In the HPLC analysis, examining the effect of pH values of the mobile phase on the capacity factor (k′) of nicardipine revealed a method for selecting a critical k′ value of nicardipine to eliminate interfering peaks near the peak specific to the analyte. This method for quantification of nicardipine in human plasma samples was suitable for studying the pharmacokinetic profile of nicardipine administered as an intravenous bolus to cardiac surgical patients.     

Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous two-phase systems

The kinetically controlled synthesis of cephalexin in aqueous two-phase systems was studied, using immobilized penicillin acylase, 7-amino 3-desacetoxycephalosporanic acid as nucleophile and phenylglycine methyl ester as acyl donor. The organic phases used were 80% (v/v) polyethyleneglycol 400 and 600 and the aqueous phase was 2.5 M (NH₄)₂SO₄. 7-amino 3-desacetoxycephalosporanic acid and cephalexin partition coefficients were determined at pH 7.4 and 7.8, at 14 °C and 20 °C. Highest partition coefficient for cephalexin was obtained for polyethyleneglycol 400–(NH₄)₂SO₄ at pH 7.4 and 20 °C, while the lowest partition coefficient for 7-amino desacetoxycephalosporanic acid was obtained in the same system at pH 7.8 and 14 °C. No significant effect of pH was observed on conversion yield and productivity of cephalexin synthesis; however, higher values were obtained with polyethyleneglycol 400 as organic phase. Higher conversion yields with both biphasic systems were obtained at the lowest temperature, where product hydrolysis was lower; volumetric productivity was higher for the fully aqueous medium (control), being higher at 20 °C. All parameters of synthesis were improved at higher substrates concentrations, obtaining conversion yields of 78.2% and 65.4%, with 60 mM 7-amino desacetoxycephalosporanic acid for the polyethyleneglycol 400–(NH₄)₂SO₄ system and the control, respectively.     

An inducible hydrolase from Mortierella isabellina (basidiomycetes). The deacylation of (+)-usnic acid

An inducible enzyme catalysing the hydrolysis of (+)-usnic acid to (+)-2-desacetylusnic acid and acetic acid has been purified 150-fold from the mycelium of Mortierella isabellina grown in the presence of (+)-usnic acid. Purification was achieved by treatment with protamine sulfate, (NH₄)₂SO₄ fractionation, negative adsorption on alumina Cγ gel and hydroxylapatite followed by chromatography on DEAE-cellulose and Sephadex G-200. The elution pattern from a Sephadex G-200 column indicated a MW of ca 7.6 × 10⁴ for the enzyme. The apparent K_m value for (+)-usnic acid at the pH optimum (pH 7) was 4.0 × 10^?5 M. The enzyme was specific for (+)-usnic acid and inactive towards (?)-usnic acid, (+)-isousnic acid or certain phloracetophenone derivatives. Its activity was enhanced in the presence of divalent metal ions such as Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺ and Zn²⁺.     

Crystallographic study of single crystals of mitochondrial coupling factor (BF1) from beef heart

Beef heart mitochondrial coupling factor (BF₁) was crystallized from 0.1 M Tris-PO₄, pH 7.8, containing 1 mM EDTA, 4 mM ATP and 1.85 M (NH₄)₂SO₄, at 22°C. Single crystals were obtained different from those reported by Spitzberg and Haworth (Biochim. Biophys. Acta 492, 237–240, 1977). The X-ray diffraction pattern reveals an orthorhombic lattice with a = 150 Å, b = 132 Å and c = 180 Å and, according to the absence of reflection, a space group of C222₁. The crystal density was determined to 1.19 g·ml^?1 and, assuming a molecular weight of 350,000 for BF₁, there is only one half of the BF₁ molecule in the asymmetric unit cell.     

Resolution of human mercapt- and nonmercaptalbumin by high-performance liquid chromatography

Gel-exclusion high-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on PGP 2000 column (0.10 M sodium phosphate buffer, 0.30 M NaCl, pH 6.86) showed at least two peaks, the principal component corresponding to human mercaptalbumin (HMA) and the second one to human nonmercaptalbumin (HNA). Mechanism for the separation of HMA and HNA might be due to weak resin-HSA interaction. HPLC analysis of bovine plasma albumin (BPA) showed a single peak on PGP 2000 column. The elution volume of HSA was larger than that of BPA, resulting in a clear resolution of HSA and BPA.     

Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2

The aim of this investigation was to test the hypothesis that elevation of intracellular pH would inhibit iron uptake by reticulocytes. The experiments were performed with rabbit reticulocytes and iron bound to rabbit transferrin. Incubation of the cells with NH₄Cl, (NH₄)₂CO₃, CH₃NH₂ and (CH₃)₂NH was used in an attempt to increase intracellular pH. These substances were all found to inhibit iron uptake by reticulocytes. The mechanism of action of NH₄Cl and CH₃NH₂ was investigated in detail. Similar results were found with both reagents. They inhibited iron uptake in a concentration-dependent manner, but produced a small increase in the cellular uptake of transferrin. The onset of action was rapid and the effect was reversible. There was no decrease in the number of transferrin-binding sites per cell and their apparent affinity for transferrin increased slightly, while the efficiency of iron removal from transferrin per binding site diminished greatly. The rate of transferrin release from reticulocytes was unaffected. NH₄Cl did not affect the rate of iron release from transferrin in a cell-free system. Incubation of reticulocytes with 10 mM NH₄Cl or CH₃NH₂ was found to produce an increase in intracellular pH of 0.05—0.15 pH units. The intracellular pH determined by used of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione was significantly higher than that obtained with the weak base (CH₃)₂NH. By transmission electron microscopy it was shown that reticulocytes treated with NH₄Cl or CH₃NH₂ have enlarged intracellular vesicles. The results are considered to support the hypothesis that iron release from transferrin in reticulocytes occurs as a result of protonation of the transferrin within intracellular vesicles. According to this hypothesis, weak bases such as NH₃ and CH₃NH₂ inhibit iron release by neutralizing H⁺ within the vesicles.     

Evidence of the importance of pH and salt concentration on column chromatography with Sepharose gel filtration in separation of proteins.

Gel filtration chromatography with Sepharose agarose gel has been widely applied in the purification of enzymes because of its capability to separate macromolecules according to molecular size. Although a wide range of pH and salt concentrations have been suggested for its use, we have found that the selectivity, or efficiency, of separation is strongly affected by the pH and salt concentrations actually used. Separation is best at neutral pH with low salt concentrations. Increasing the molarity of the buffer or salt content (such as ammonium sulfate) in the protein sample will either broaden protein peaks resulting in poor separation or displace the peaks to a position of much lower apparent hydrodynamic volume. Rabbit plasma monoamine oxidase (MAO), a protein of 150,000 MW, when combined with 1.3 m (NH₄)₂SO₄ at pH 5.4, was found to be retained in Sepharose 6B column until the very end and elute with ammonium sulfate molecules. This behavior was attributed to severe morphological changes on the gel surface at acidic pH leading to a loss of selectivity. Evidence for this interpretation is provided by parallel experiments with Sephadex columns under identical conditions which excludes the possibility of dissociation of MAO into subunits and by scanning electron microscopy which demonstrates the change of surface morphology of the gel. The necesslty of a careful selection of optimum conditions for Sepharose gel chromatographic separation is therefore suggested.     

Selective separation of human peripheral platelets,granulocytes and lymphocytes by surface affinity chromatography

Selective separation of human peripheral platelets, granulocytes and lymphocytes was investigated by column liquid chromatography using methoxyethoxymethyl (MEM) bonded-phase columns (25 × 0.9 cm I.D.). Isotonic solutions containing mono- and disaccharides, methyl-α-d-pyranosides and a physiological saline at pH 7.4 were used as the mobile phase. Granulocytes and lymphocytes were separated on the MEM-Cellulofine GH-25 column by elution with 0.3 M d-mannose solution. The isolation of platelets and lymphocytes from human leukocyte-rich plasma was performed with a MEM-Sephadex G25 column and elution with 0.27 M sucrose solution. On the same column platelets could also be collected selectively by elution with 0.31 M methyl-α-d-mannoside at the high recovery of 100%. The isolated cells were viable for more than 90%.     

Influence of pH upon the Warburg Effect in Isolated Intact Spinach Chloroplasts: I. Carbon Dioxide Photoassimilation and Glycolate Synthesis

The influence of pH upon the O₂ inhibition of ¹⁴CO₂ photoassimilation (Warburg effect) was examined in intact spinach (Spinacia oleracea) chloroplasts. With conditions which favored the Warburg effect, i.e. rate-limiting CO₂ and 100% O₂, O₂ inhibition was greater at pH 8.4 to 8.5 than at pH 7.5 to 7.8. At pH 8.5, as compared with 7.8, there was an enhanced ¹⁴C-labeling of glycolate, and a decrease of isotope in some phosphorylated Calvin cycle intermediates, particularly triose-phosphate. The ¹⁴C-labeling of starch was also more inhibited by O₂ at higher pH. The enhanced synthesis of glycolate during ¹⁴CO₂ assimilation at higher pH resulted in a diminution in the level of phosphorylated intermediates of the Calvin cycle, and this was apparently a causal factor of the increased severity of the Warburg effect.     

Physicochemical properties of salt-soluble,unsheared chromatin

A chromatin fraction, which can reproducibly be extracted from rat liver nuclei at moderate salt concentration (0.1 M (NH₄)₂SO₄, 0.1 M Tris-HCl, 2 mM MnCl₂, pH 7.9), was analyzed with regard to changes of its molecular weight in the range of (NH₄)₂SO₄ concentrations between 0.1 M and 0.4 M. With the transition from 0.1 M to 0.2 M (NH₄)₂SO₄ histone H1 is released and the molecular weight obtained from both sedimentation-viscosity and light scattering is reduced by approximately one-half. A spatial expansion of the resulting half-molecules is observed with further increasing salt concentration. On the basis of these results a double-fibrillar structure of this chromatin fraction is proposed.     

Correlation between photosynthesis and the transthylakoid proton gradient

In isolated intact chloroplasts, maximal rates of photosynthetic O₂ evolution (in saturating HCO^?₃) are associated with a critical transthylakoid proton gradient as a result of the stoichiometric consumption of 2 mol NADPH and 3 mol ATP/mol CO₂ fixed. Studies with the fluorescent probe 9-aminoacridine reveal that in the illuminated steady state the critical ΔpH is 3.9.CO₂-dependent O₂ evolution is inhibited by increases of 0.1–0.2 in ΔpH that occur when catalase is omitted from the medium, NO^?₂ is included as an electron acceptor, or when chloroplasts are illuminated under low partial pressures of O₂. Low concentrations of antimycin (0.33 μM) or NH₄Cl (0.33 mM) decrease ΔpH and relieve this inhibition of electron flow. The energy transfer inhibitor quercetin lowers the high ATP/ADP ratio associated with these conditions, but does not lower ΔpH or relieve the inhibition.A decrease of ΔpH below 3.9 by weaker illumination, millimolar levels of NH₄Cl or micromolar levels of antimycin, results in lower rates of photosynthesis owing to limitation by the phosphorylation rate.These findings show that in absence of rate limitation by the carbon cycle, the extent of thylakoid energization is related to the ratio of ATP to NADPH production and in turn, the rate of CO₂ assimilation.     

Isolation of antibodies to steroid hormones by affinity chromatography on antigen-linked sepharose: An efficient electrophoretic elution procedure

An electrophoretic elution procedure of antibodies retained on affinity columns is described. It afforded a 60% recovery of the binding activity of a high affinity (Ka ～ 10¹⁰ M^?1) antiserum to 5α-dihydrotestosterone retained on antigen-linked Sepharose 4B affinity columns. These purified unbound antibodies, (Ka ～ 10¹⁰ M^?1) when applied again on identical antigen-linked affinity columns, were all retained and totally recovered after a new electrophoretic elution. Comparable results were obtained by elution with 1M NH₄OH.The residual 40% binding activity remaining on the antigen-linked Sepharose gel after electrophoretic elution was totally recovered by elution with an excess of 5α-dihydrotestosterone. It corresponded to antibodies of higher affinity (Ka ～ 10¹¹ M^?1). On the other hand the residual 40% fraction of antibodies resistant to NH₄OH elution was denaturated.     

Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A x W182E) : Two Molecular Forms of the Enzyme

The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N₂ and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0°C and for several months at −80°C. The enzyme was purified using (NH₄)₂SO₄ fractionation, brushite-hydroxyl-apatite chromatography and blue-sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO₃. About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO₂⁻ per minute per milligram protein). A sequential elution with NADH followed by KNO₃ (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO₃⁻-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed.     

Preparation of Large Oligonucleotides by RNase T1 Partial Hydrolysis of MS2 Virus Treated with Succinic Nhydride

Intact MS2 virus was reacted with succinic anhydride to modify the protein coat and then treated with RNase Tl to obtain controlled hydrolysis of the viral RNA. Viral protein and enzyme were removed by phenol extraction. The RNA fraction was adsorbed on DEAE-cellulose and eluted stepwise with 0.1, 0.5, and 1.0 M NH₄HCO₃, pH 8.6. tRNA^arg, used as a marker, was eluted in the 1.0 M NH₄HCO₃ fraction. The oligomers in the 1.0 M fraction isolated from the viral derivative were further examined by paper chromatography, polyacrylamide gel electrophoresis, and sucrose gradient cestrifuga-tion. Yields of the large oligomer fraction as defined by elution with 1.0 MNH₄HCO₃ from DEAE-cellulose ranged from 51–86% of the amount applied.     

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.