Procion Red Research Articles

Amylase substrate based on fluorescence energy transfer

A fluorigenic substrate for measuring α-amylase (E.C. 3.2.1.1) activity was prepared by double labeling soluble starch with 5-(4,6-dichlorotrizain-2-yl)aminofluorescein and Procion Red MX8B. Because the absorption spectrum of Procion Red MX8B overlaps the fluorescein emission spectrum, Procion Red efficiently quenches fluorescein emission when it is closer than the critical radius for fluorescence energy transfer. When amylase catalyzes cleavage of a starch molecule between a fluorescein and a Procion Red MX8B, the distance between the two labels increases and the degree of quenching decreases. The rate at which the fluorescence intensity increases is proportional to amylase activity. To maximize the sensitivity it is critical to maximize the amount of Procion Red MX 8B coupled to the starch and to use a high-precision spectrofluorimeter which can measure a small rate of increase in fluorescence above a large constant background.

Reaming versus non-reaming in medullary nailing: Interference with cortical circulation of the canine tibia

Medullary nails have only been used with reluctance in the treatment of open fractures since reaming interferes with the cortical circulation. The technique of interlocking offers the interesting possibility of inserting a relatively stiff nail which does not necessarily require reaming. The effects of reaming prior to nail insertion upon the cortical circulation was investigated in the canine tibia. Intravital staining with procion red was used to trace areas of disturbed circulation. Nail insertion without reaming provided clear advantages for the bone's blood supply, while reaming disturbed perfusion in two-thirds of the cortical area and regionally extended through the entire thickness of the cortex. The disturbance without reaming was limited to the inner layer of the cortex and involved only one-third of the cortical cross-section.

P-319 - Ca2+-dependent interaction of annexins from bovine lung with Procion Red HE-3B

P-319 - Ca2+-dependent interaction of annexins from bovine lung with Procion Red HE-3B

Purification and properties of soluble hydrogenase from the cyanobacterium Anabaena cylindrica

Two soluble hydrogenase activities were separable from cell extracts of the cyanobacterium Anabaena cylindrica, one detectable by the tritium exchange assay, the other having a relatively low tritium exchange activity but catalyzing methyl viologen-dependent hydrogen formation. Their molecular weights, by gel filtration chromatography, were 42,000 and 100,000, respectively. The two hydrogenase activities were differentially inhibited. The methyl viologen-dependent activity has been purified to homogeneity from cells in which the enzyme was induced by gassing the growing cells with N 2/H 2/CO 2 (95.7%/4%/0.3%, v/v/v). The procedure involved French pressure cell disruption of the cells, differential precipitation with ZnCl 2, heat treatment (50 °C), and lyophilization of the heat-step supernatant. It was then subjected to DEAE-Sephacel chromatography, dye-ligand chromatography on Procion Red, and HPLC anion exchange on QMA-Accel. Polyacrylamide gel electrophoresis on both native and denaturing gels revealed two peptides with M r's 42,000 and 50,000. The 42,000 protein alone catalyzed tritium exchange activity; both proteins appeared to be necessary for the methyl viologen activity. The native enzyme appears to be a readily dissociable dimer of two nonidentical subunits, one of which contains the hydrogen binding site and the other providing the ability to utilize electrons from a reductant for hydrogen formation.

Interaction of lactate dehydrogenase with structurally related triazine dyes using affinity partitioning and affinity chromatography

Affinity partitioning in aqueous two-phase systems consisting of dextran and dye-liganded polyethylene glycol was employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle (E.C. 1.1.1.27) with Procion Red HE-3B and four structurally related derivatives of this dye in order to follow the significance of the terminal rings of Procion Red HE-3B for the strength of interaction. The study revealed that the arrangement of the two 1-amino-8-naphthol-3,6-disulphonic acid rings seems to be a prerequisite for the interaction of azonaphthol dyes with LDH. The negatively charged sulfonic acid group at the terminal rings of Procion Red HE-3B enhances the affinity of the ligand for LDH significantly. The removal of this sulphonic acid group or splitting off the complete terminal rings decreases the affinity to LDH and improves the competitive effect of NAD +. The results of affinity partitioning are compared with those of affinity chromatography and kinetic data. The usefulness and the choice of parameters of affinity partitioning as an analytical tool to predict the chromatographic behaviour of dye ligands are discussed.

Lyophilized Clostridium perfringens 3α- and Clostridium bifermentans 7α-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis

Preparations of 3α-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3α-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3α-hydroxysteroid dehydrogenase and 7α-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20–100 μM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3α-hydroxysteroid dehydrogenase and showed also that 7α-hydroxyl groups were independently quantified by the 7α-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purifiedpreparations of Pseudomonas testosteroni 3α-hydroxysteroid dehydrogenase, C. perfringens 3α-hydroxysteroid dehydrogenase, Escherichia coli 7α-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7α-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3α- and C. bifermentans 7α-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.

Crystallization of recombinant human interleukin 1β

The gene for the fully processed form of human interleukin 1β was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in E. coli. The protein produced in E. coli. was purified to homogeneity by standard chromatographic methods, including adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 FPLC column, and anion exchange chromatography on QAE Sepharose. The result is a biologically active protein, rIL-1β, that migrates on two-dimensional gels as a single spot with a pI of 6.5 ± 0.2 and a molecular mass of 17, 500 daltons. Crystals of rIL-1β have been produced from concentrated solutions of the protein by ammonium sulfate precipitation. The crystals are tetragonal, have space group P41 or its enantiomer, have lattice constants of a = 58.46(1) Å and c = 77.02(3) Å, and scatter to at least 2 Å resolution. A structure determination ba these crystals is underway.

Purification and characterization of phytase from cotyledons of germinating soybean seeds

Soybean phytase ( myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 × 10 4 m −1 cm −1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250–300 m m. The enzyme possessed a high affinity for phytic acid (apparent K m = 48 μM), and was strongly inhibited by phosphate (apparent K i = 18 μM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5–4.8 and 55 °C, respectively.

Affinity chromatography with triazine dyes immobilized onto activated non-porous monodisperse silicas

Non-porous monodisperse silicas with a particle diameter of 2.1 μm were modified with different silanes for immobilization of various triazine dyes including Procion Red HE3B, Procion Red MX5B, and Cibacron Blue F3GA. Lactate dehydrogenase and malate dehydrogenase from different species and aldehyde reductase from rat brain were purified by affinity elution using the substrate of the enzyme and NADH. With Cibacron F3GA the selectivity for NADH-dependent enzymes was higher than with the two Procion dyes. The utility of these immobilized triazine dye systems on non-porous silica supports for the rapid separation of Cohn Fraction III plasma proteins, including plasminogen, is also described.

Crystallization of purified recombinant human interleukin-1 beta.

The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution. A structure determination based on these crystals is under way.

Purification of 6-phosphogluconate dehydrogenase from Acer pseudoplatanus L. using immobilised procion red HE-3B

The effect of the support matrix on the capacity of immobilised Procion Red HE-3B for a higher plant NADP +-dependent dehydrogenase is examined. A method is presented for the purification of 6-phosphogluconate dehydrogenase (E.C. 1.1.1.44.) from Acer pseudoplatanus L. (sycamore) which includes chromatography on Procion Red-Sepharose. The enzyme was purified 380-fold with 40–50% recovery and a final specific activity of 80 units per mg protein. Preliminary evidence was obtained for the existence of isoenzymes of 6-phosphogluconate dehydrogenase in Acer pseudoplatanus L.

Novel cationic triazine dyes for protein purification

The effectiveness of a new immobilized cationic triazine dye was investigated alongside two new amphoteric triazine dyes and two well known anionic triazine dyes, Procion Red H-3B and Procion Blue H-B, as chromatographic media for binding four familiar proteases-trypsin, chymotrypsin, thrombin and carboxypeptidase-B-as well as a typical oxidoreductase, lactate dehydrogenase, and human serum albumin. The new affinity adsorbent, CL-Sepharose-immobilized Cationic Dye, specifically binds trypsin-like proteases such as trypsin, thrombin, and carboxypeptidase-B, but none of the other proteins tested. In contrast, the amphoteric and anionic immobilized dyes bind all the other proteins tested in a similar fashion. The specificity of the cationic dye was exploited in the resolution of trypsin and chymotrypsin from a crude activated bovine pancreatic extract. The procedure described here affords trypsin with specific activity of 7400 units/mg with a 79% overall yield in a single step. The immobilized cationic dye, unlike previously reported adsorbents for trypsin, is inexpensive, readily synthesized, and displays a workable capacity of 4000 trypsin units or 0.55 mg protein/g moist weight gel (1.2 mumol dye/g moist weight gel) from a crude bovine pancreatic extract and, thus, is potentially amenable to process-scale operations.

Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli.

An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an AvaII fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis.

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies.

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.

Interaction of Cibacron Blue F3G-A and Procion Red HE-3B with sheep liver 5,10-methylenetetrahydrofolate reductase

Cibacron Blue F3G-A, a probe used to monitor nucleotide binding domains in enzymes, inhibited sheep liver 5,10-methylenetetrahydrofolate reductase competitively with respect to 5-methyltetrahydrofolate and NADPH. TheKi values obtained by kinetic methods and theKd value for the binding of the dye to the enzyme estimated by protein fluorescence quenching were in the range 0.9–1.2 μM. Another triazine dye, Procion Red HE-3B interacted with the enzyme in an essentially similar manner to that observed with Cibacron Blue F3G-A. These results as well as the interaction of the dye with the enzyme monitored by difference spectroscopy and intrinsic protein fluorescence quenching methods indicated that the dye was probably interacting at the active site of the enzyme by binding at a hydrophobic region.

Purification and physical-chemical characterization of hepatic stimulator substance†

Hepatic stimulator substance is a liver growth stimulator derived from the hepatocyte cytosol of weanling or regenerating adult rat livers. The present paper reports the almost 9,000-fold purification of hepatic stimulator substance with an approximately 100,000-fold increase in specific growth stimulator activity. Purification steps included heating at 95 degrees C for 15 min, 40% cold ethanol precipitation, passage over Procion Red HE3B, DEAE cellulose and Sephadex G75 columns and gel filtration and reverse-phase fast protein liquid chromatography techniques. As little as 27 ng per ml of the purest material produced a 2-fold stimulation in the standard HTC cell activity assay. Further studies indicate that hepatic stimulator substance is a highly negatively charged protein and that disulfide bonds or a complex tertiary structure are not essential to its activity. Hepatic stimulator substance is stable over a wide range of pH's and temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver stain revealed 1 major band at 12,400 daltons and 1 minor band at 17,500 daltons.

Eosinophil Chemotactic Lymphokine Produced by Spleen Cells of <i>Schistosoma japonicum</i>-lnfected Mice

Two distinctive types of eosinophil chemotactic lymphokines (ECF-L) directed against eosinophils obtained from bone marrow (BM-Eo) or from peritoneal cavity (PEC-Eo) were isolated from the media conditioned by spleen cells from mice infected with <i>Schistosoma japonicum</i> by a combination of anion-exchange chromatography on DE52, affinity chromatography on Procion Red agarose, and high-performance liquid chromatography. The molecular weight of ECF-L directed against BM-Eo was 10,000 and that against PEC-Eo was 46,000 by SDS-PAGE analysis. After isolation, selectivity of the two distinct ECF-L was examined using BM-Eo, PEC-Eo and circulating Eo. ECF-L directed against BM-Eo could attract only BM-Eo, whereas that directed against PEC-Eo could attract not only PEC-Eo but also circulating Eo. Synthetic ECF-A was chemotactic against both BM- and circulating-Eo but not against PEC-Eo. When BM-Eo were cultured with ECF-L selective to PEC-Eo, they became reactive to ECF-L selective to PEC-Eo, whereas ECF-L selective to BM-Eo did not have such effect. These results suggest that ECF-L selective to PEC-Eo is not simply a chemo-attractant but also is important in eosinophil differentiation.

Effects of organic solvents on the partitioning of enzymes in aqueous two-phase systems

Organic solvents (ethylene glycol, glycerol, dimethyl sulphoxide, dimethylformamide, dioxane, methanol and propanol-2, as well as sucrose and urea) have been included in aqueous two-phase (liquid—liquid) systems comprised of water, dextran and poly(ethylene glycol). The concentration of the organic solvent was in most cases 20% (w/w). The influence of these solvents on the phase-forming properties, the volume ratio, the freezing point and the partitioning of a polymer-bound ligand, Procion Red HE-3B poly(ethylene glycol), has been studied. The partition coefficients for alkaline phosphatase decrease with ethylene glycol, glycerol, sucrose and urea (factors of 0.25–0.5), but increase with the other substances (factors of 1.2–1.6). The temperature effects on the partitioning of alkaline phosphatase from calf intestine as well as of phosphofructokinase from yeast in systems containing ethylene glycol have been studied and compared with partitioning in standard systems, not containing solvents. The possible uses of the above systems for partitioning studies of enzymes are discussed.

The fractionation of soil polysaccharide by electrofocusing

SUMMARYSoil polysaccharide isolated from alkali extracts of Countesswells and Insch series soils has been submitted to electrofocusing with ampholyte in dextran gel. To aid detection, polysaccharide which had been dyed with either Procion Red or fluorescein or labelled 14C by prior incubation of the soil with 14C‐labelled substrates, was also used.After electrofocusing, polysaccharide was distributed in the gel over the entire range of pH used, 1.9–10, with the greatest concentration occurring within the pH 1.9–4 region. The distribution was partly the result of adsorption and diffusion. Re‐electrofocusing of material isolated from particular regions of the bed located most of it in the original position, although considerable dispersion occurred, particularly with the less strongly charged material and there was evidence for association/dissociation reactions. This was also observed in a medium containing 8 M urea.The proportion of uronic acid was greatest in the polysaccharides fractions from the pH 3.0–4.0 region of the bed. The increased acidity of the fraction nearest to the anode appeared to relate to the presence of other organic acids, such as succinic, fumaric and levulinic acids. No difference could be detected between the separation of polysaccharide from the two different soils or between the labelled polysaccharides derived from an incubation of 14C‐glucose with soil and those from an incubation of 14C‐plant material.

Sexual dimorphism in immunoneutralization of bioactivity of rat and ovine inhibin.

Inhibin was partially purified from bovine follicular fluid using chromatography on immobilized Procion Red 3B and anion-exchange chromatography. Ovariectomized Texel ewes were immunized against the inhibin-containing fraction from the Procion Red 3B column and the immune response was subsequently boosted with similar fractions or with the preparation obtained from the anion-exchange column. The potencies of the resulting antisera were evaluated in an in-vitro bioassay system for estimating inhibin activity, using dispersed rat pituitary cells. The antisera were found to inhibit the bioactivity of inhibin preparations from ovarian follicular fluid of bovine, porcine, ovine or human origin, as well as inhibin activity in ovine testicular lymph and rete testis fluid, in culture media from rat granulosa and rat Sertoli cells and in homogenates of rat ovaries and testes. These results indicate that the inhibin molecules from several species contain a common bioactive moiety. The results also showed that the antiserum was more effective in neutralizing inhibin activity from ovarian than from testicular sources in both sheep and rat, indicating a sex-related difference in the inhibin molecules within a species.