The acridine dyes: Their purification,physicochemical, and cytochemical properties

Summary The present investigation was designed to allow a critical comparison of the cytochemical behaviour of commercially available acriflavine dye samples and pure acriflavine and proflavine dyes, regarding their application in automated cell analysis. Thin layer chromatography, NMR-spectroscopy and mass-spectrometry were applied for the identification of the dye composition.This study includes (1) a column chromatographic technique for the purification of larger dye quantities, (2) the investigation of the photodecomposition of different dye samples, and (3) the evaluation of the influence of various acriflavine/proflavine dye concentrations (1.6·10^–3–4·10^–6 mol/l) on to the emission spectrum of stained unhydrolyzed and hydrolyzed chicken erythrocytes.The commercially available acriflavine dye samples showed a much higher reduction in fluorescence intensity than the pure dyes, whereby proflavine faded less than acriflavine. Photodecomposition is markably influenced by dye impurities. Fluorescence emission spectra were registered at various acriflavine and proflavine dye concentrations for unhydrolyzed and hydrolyzed chicken erythrocytes in order to investigate the dye-dye interaction and the behaviour of the cellular DNA-dye complex. Proflavine showed a similar spectral behaviour as acriflavine. The dye concentration-dependent spectral behaviour of the DNA-dye complex of these fluorochromes seems to be a very critical factor. A comparison of quantitative fluorescence measurements can only be performed by staining cells with the same dye quality, because automated cytology requires reproducible information of cells in machinesensible terms.This investigation was supported by a grant from the Bundesministerium für Forschung und Technologie (01 VH 065)     

The azure dyes: their purification and physicochemical properties. II. Purification of azure B.

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or polychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polymeric adsorbent (XAD-2) the acetate-formate anions are exchanged in chloride. Regeneration of both columns is possible: KMnO4, Na2S2O4 and water are run through column A; 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

The metachromasia of fluorescence of acridine dyes

Summary 1. Fluorescence- and absorption spectra of a number of acridine dyes were measured at several concentrations, in different solvents and at various pH and temperatures.2. In aqueous solutions metachromatic shifts are visible with all dyes — except acridine-yellow — with increasing concentrations, even when the dye is present as di- or trivalent ions in strongly acid solutions.3. Under conditions where reabsorption of the fluorescent light is excluded: completely separated fluorescence and absorption spectra, or measurement of fluorescence in capillaries with 0.8 mm internal bore, metachromatic effects are absent.4. Reasons are given to consider the hypothesis of specific aggregation (formation of dimeric dye-particles) as doubtful. In the case of the acridine dyes the optical properties of the monomeric dye ions are sufficient to explain the metachromatic shifts.With 10 Figures in the Text     

Pig liver fatty acid synthetase: purification and physicochemical properties.

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 mm dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of S^o_20,w = 13.3 S and D^o_20,w = 2.60 × 10^?7cm²/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10^?7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of $\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{s}\text{D}\text{) = 478,000}$ and sedimentation equilibrium data yielded a value of M_w = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.     

Isolation, purification and physicochemical properties of glutamin-asparaginase from Pseudomonas boreopolis 526

A new homogeneous enzyme which is capable of catalyzing the hydrolysis of both glutamine and asparaginase has been purified from extracts of Pseudomonas boreopolis 526 by the improved method. Purification involves few stages. The ratio of glutaminase to asparaginase activity is approximately 1.5:1.0. The enzyme is stable on storage and has a wide pH optimum of action (6-8.5). The molecular weight is about 134 000-145 000 D and the subunit molecular weight is about 34 000 D. No free SH-groups have been detected in the enzyme molecule.     

The ninth component of human complement: purification and physicochemical characterization

A procedure for the isolation of the human complement (C) protein C9 is described. The procedure allowin. The purified protein has the electrophoretic mobility of an alpha-globulin, and is a single polypeptide chain with a m.w. of 71,000. No impurities were detected either on gel electrophoretic or immunochemical examination. C9 is a glycoprotein containing 7.8% carbohydrate, and in terms of residues per mole, 3.0 glucosamine, 17.6 neutral hexose, and 7.4 sialic acid. Its amino acid composition is typical of a globular serum protein. Upon automated Edman degradation of reduced and alkylated C9, no amino acid residues were released, suggesting a blocked N-terminus. The concentration of C9 in normal human serum is 58 +/- 8 microgram/ml. A high titer rabbit antiserum was produced and employed to immunochemically deplete serum of C9. The CH50 of the C9-depleted serum was identical to that of whole human serum; however, membrane fragments of erythrocytes lysed by C9-depleted serum lacked the typical ultrastructural C lesions, which constitute the dimeric membrane attack complex.     

Carbon monoxide dehydrogenase from Methanosarcina barkeri. Disaggregation, purification, and physicochemical properties of the enzyme

Carbon monoxide dehydrogenase from acetate-grown cells of Methanosarcina barkeri exists in a high molecular weight form (approximately 3 X 10(6)) under conditions of high ionic strength but is converted to a much smaller form by dialysis. The enzyme was purified by a procedure which exploits isolation of the aggregated form by gel filtration and subsequent dissociation. Following this, the enzyme was purified to within 92% of homogeneity by chromatography on phenyl-Sepharose and finally on hydroxylapatite. Due to the extreme oxygen lability of the enzyme, the entire procedure was carried out within the anaerobic laboratory at the National Institutes of Health. The enzyme has an alpha 2 beta 2 oligomeric structure composed of subunits with molecular weights of 19,700 and 84,500. The amino acid compositions of the individual subunits were determined. Analysis of the metal content by plasma emission spectroscopy indicated 1.3 +/- 0.3 (n = 4) nickel and 15.6 +/- 5.6 (n = 5) iron per mol of alpha 2 beta 2. The enzyme did not contain significant amounts of cobalt or molybdenum. Ferredoxin, FAD, FMN, 2,3,5-triphenyltetrazolium chloride, methyl viologen, and phenazine methosulfate served as electron acceptors; however, the enzyme failed to reduce NAD+, NADP+, or the 8-hydroxy-5-deazaflavin factor F420. The optimum pH was between 7 and 9. The apparent Km for methyl viologen was 7.1 mM, whereas the value for 2,3,5-triphenyltetrazolium chloride was below 0.5 mM. Strong inhibition was observed by oxygen and cyanide. Inactivation by glyoxaldehyde required enzymatic turnover which suggested that a reactive group was formed, or exposed, on an enzyme intermediate in catalysis. A high degree of thermostability was noted. Carbon monoxide, however, rendered the enzyme more susceptible to temperature inactivation.     

The effect of acridine dyes on the molecular structure of DNA]

DNA--acriflavin complexes have been investigated by the methods of flow birefringence and viscometry. The intrinsic viscosity and the optic anysotropy of the complex increase with the increasing quantities of binding dye. Experimental data are treated on the basis of different models of binding. At high ionic strength (mu = 0,1) one type of binding takes place which is described by the intercalation model. In this case the thermodynamic rigidity of DNA-molecule within the complex is proportional to "r". In solutions of low ionic strength (mu = 0,001), two types of DNA-acriflavin binding occur: intercalation and external binding. At low ionic strength, the spectrophotometric titration technique is shown to give a reduced value of "r".     

Isolation, purification and physicochemical properties of pesticin I from cells of Yersinia pestis strain EV

Pesticin I has been isolated and purified from Y. pestis strain EV. The homogeneous preparation of Pesticin has been shown to be monomer protein with a molecular weight of 65000 daltons, having three immunologically identical alpha-, beta- and gamma-forms with different isoelectric points. The amino acid composition of Pesticin I is presented. Rabbit anti-serum to the beta-form of the preparation of Pesticin has been obtained.     

Large-scale purification, crystallization and some physicochemical properties of extracellular guanyl-RNases C2 and Pch1]

The purification of RNase C2 from 76.5 1 of Asp. clavatus cultural fluid and RNase Pch1 from 160 1 of Pen. chrysogenum 152 A cultural fluid was described. 1150-fold purification of RNase C2 was attained by precipitation with ammonium sulfate, ion-exchange chromatography and rechromatography on DEAE-cellulose, gel chromatography on Sephadex G-75, and crystallization from diluted acidic buffer. During the preparation of RNase Pch1 additional chromatography on CM-cellulose was used before crystallization, the purification being 2220-fold. It was obtained 600 mg RNase C2 and 900 mg RNase Pch1. Some physico-chemical properties of crystalline RNases were studied.     

The use of dyes in protein purification.

The role of the matrix, ligand and linking mechanism in affinity chromatography is discussed, special emphasis being placed on the use of dyestuff molecules as ligands. Current knowledge of dye-protein interactions is outlined and problems arising from the use of conventional textile dyes as ligands are considered. Work on the synthesis of novel dye-like molecules designed specifically for affinity chromatography is reviewed. This is seen as leading to the development of improved affinity systems capable of advancing the utility of affinity chromatography in protein purification.