Search

A convenient and relatively efficient technique was developed for the routine measurement of radioactivity distribution on one-dimensional paper chromatograms containing low amounts of H3- and C14-labeled compounds which are insoluble in the scintillation solvent toluene. A pilot strip, 2 cm wide, was cut out from the chromatogram and divided into 3 4 cm bands along the length of the chromatostrip. Each band was placed in a standard 20-ml screw-top glass vial. Methanol (0.2 ml) was added and the contents were shaken occasionally for 2 or 3 min. Both the methanol and filter paper band were flooded with 10 ml scintillation fluid and the contents were counted. This technique was found to be more satisfactory in terms of efficiency and reproducibility than several other methods tested.

A simple procedure for obtaining good infrared spectra of compounds eluted from paper chromatograms has been described. As little as 25 μg of many phenolic acids may be isolated, characterized, and identified from paper chromatograms.

Summary A paper chromatographic procedure is described for detecting dithioacetals of sugars. A 1% aqueous solution of potassium permanganate was used as the spraying reagent, by which the sugar dithioacetals were detected as the brown spots on paper chromatogram. A series of developing solvents was investigated, and some of them were found useful for separating 2-amino-2-deoxy- d -glucose diethyl dithioacetal from 2-amino-2-deoxy- d -galactose diethyl dithioacetal, and both the diethyl dithioacetals from uronic acid diethyl dithioacetals.

The new paper chromatographic system, isopropyl ether-ethylene glycol system, provides an improved and faster separation of steroids, particularly estrogens in paper chromatograms. The mobilities of a number of steroids are listed.

The problems confronting the chemist in control of water treatment for an industrial boiler plant are very complex and dictated to a great extent by the pattern of plant operation. An interesting field of study is presented, particularly where the plant is modern in design and very extensive in the service it provides. The author has concerned himself principally with the problems of water supply and treatment at Aylesford Mills, as these are very typical of those which occur in other large industrial undertakings. Details are given of the feed water treatment plant for the medium- and high-pressure boilers, consisting of primary sedimentation units using lime, ferrous sulphate, and alum as chemical additives, followed by a filtration stage. This section of the plant is followed by a dual system comprising three base-exchange softener units and a fully automatic demineralization plant. The interesting features of fully automatic regeneration of the demineralization plant are discussed, together with the author's experience in operating this plant. Emphasis is placed on high standards of feed water treatment and the principles of the system adopted. Sodium sulphite and tannin are added as chemical deoxidants on medium-pressure plant. Hydrazine, together with a neutralizing amine, are used on the high-pressure boiler installation. The author concludes the paper with comments on the subject of boiler corrosion.

Chromatographic resolution of protein mixtures: egg albumin and lysozyme, and another containing insulin and protamine (Mugil cephalus) was achieved on paper strips using a several step elution with neutral salt solutions of increasing concentrations. Experimental data concerning the influence of cations and anions on chromatographic separation of proteins on paper strips are presented.

Differentiation of radioactive DNA and RNA deposited on filter paper discs can be accomplished by a relatively simple procedure. RNA can be efficiently removed by incubating the dises, impaled on pins, with 0.2 ml of 0.5 n NaOH for 90 min at 37°C. DNA can be removed after NaOH hydrolysis by treating the discs with 5% TCA for 30 min at 90°C. A correction is necessary to determine the actual amounts of DNA and RNA in order to account for the loss of DNA (13.8%) during the NaOH hydrolysis procedure.

A simple, improved scintillation counting procedure was developed for the assay of radioactive mono- and polysaccharides on paper chromatograms. Segments of chromatograms are placed in scintillation vials and soaked in water to completely elute the carbohydrate before addition of Aquasol, a xylene-based scintillation fluid. The resulting water-Aquasol solution is counted in a liquid scintillation counter. Evaluation of numerous experimental variables revealed optimal conditions for complete elution of mono- and polysaccharides with water before counting in Aquasol. The water elution-Aquasol procedure allows water-soluble substances (14C- and 3H-labeled) on paper to be assayed with increased accuracy and sensitivity (three- to fivefold improvement in counting efficiency of tritiated samples). The simplicity of the procedure allows entire radiochromatograms to be assayed readily.

Three methods for evaluation of serum lipoprotein abnormalities were compared: paper electrophoresis using buffer containing albumin, ultracentrifugation at d 1.21, and thin-layer starch-gel electrophoresis. Analyses by paper electrophoresis and by ultracentrifugation of 109 sera of patients with cholesterol levels between 78 and 1150 mg/100 ml showed that the electrophoretic procedure while not quantitative was an effective procedure for detecting and helping in classification of the type of serum lipoprotein abnormality. Paper electrophoresis is a valuable procedure in screening for lipoprotein abnormalities and can be used as a guide for additional studies. Thin-layer starchgel electrophoresis did not always give results comparable to those of the other two procedures. In study of certain sera (for example, in pigeon serum and some myeloma sera) lipoprotein subfractions were more clearly resolved by starch-gel electrophoresis than by paper electrophoresis or ultracentrifugation. Ultracentrifugal and starch-gel electrophoretic techniques can be used effectively when more complete information is needed or when unusual materials are being studied.

Construction and use of a simple scanning device are described for locating small amounts of radioactivity on large uncut filter papers used for chromatography and electrophoresis. With this device the use of autoradiographs in eliminated from the original method for determining urinary hydroxyproline on the principle of isotope dilution. The modified method is described, and eight results are reported for the recovery of added nonradioactive dipeptide. Percentage of recovery varied from 84.5 to 111% with an average of 98.4%.

The separation of dog serum lipoproteins by ultracentrifugation at a density of 1.063 is hindered by the failure of the high density fraction to accumulate in the bottom portion of the centrifuge tube. This phenomenon interferes with the quantitative recovery of pure low density lipoproteins. By paper electrophoresis, a lipid-containing protein with the mobility of a β-globulin was detected in the lowest layer of the centrifuge tube. Comparison of the preparative ultracentrifuge technique with that of dextran sulfate precipitation of β-lipoproteins revealed the suit ability of the latter procedure for the quantitative separation of β-lipoproteins. The dextran sulfate β-lipoprotein precipitate was shown to be free from α-lipoprotein by paper electrophoresis.

The use of visual dye markers for high-voltage paper ionophoresis has been proposed to yield reproducible ionograms and to prevent losses of peptides during purification runs. Since the relative mobility of a dye to an amino acid or peptide is not affected by temperature fluctuations during ionophoresis in vertical Michl-type apparatus, the dye is allowed to migrate to a predetermined mark on the paper that ensures that amino acids and peptides remain on the paper. Mobility data are presented for dyes and certain amino acids at pH 2.0, 3.5, and 6.5 ionophoresis.

Ornithine decarboxylase has been measured in midterm mouse placentas by a simple inexpensive method. The reaction vessel was a special plastic scintillation vial. The 14CO2 liberated from dl -1-14C-ornithine was absorbed by a Hyamine hydroxide impregnated blotting paper disc cemented into the vial's cap. This was released into the toluene fluor of another scintillation vial by transfer of the cap after loosening the paper disc. The incubation vial was further adapted by predrilling a hole in its side to permit addition of acid to stop incubation and drive 14CO2 out of solution. This hole was kept sealed with tape. The preference of Hyamine over ethanolamine/2-methoxyethanol mixture (2:1) was established. Also the protective value of EDTA and dithiothreitol and the deleterious effect of freezing and thawing on this enzyme were studied. Mouse placentas contain a relatively large amount of ornithine decarboxylase. This method of assay should be suitable for quantification of other decarboxylases whenever suitable carboxyl-labeled substrates are available.

A paper electrophoresis method is described in which four serum lipoprotein components are separated without the use of albumin in the buffer. Tris-EDTA-boric acid buffer, Oil Red O staining solution prepared in 52.8% instead of the usual 60% alcohol, and solvent-extracted paper strips are the distinguishing features of this procedure. The system was effective not only in separating chylomicron, β- and α-lipoproteins into well-defined bands, but also in separating very low density or pre-β-lipoprotein distinctly in some samples and partially in others. The advantages of this procedure are low background staining and, in comparison to the procedure using albumin in the buffer, a sharper alpha band. This sharpened α-band gives a sharper peak in densitometric scanning. Electrophoresis could be performed for 16 hr or 4 hr. The 4-hr electrophoresis run produced electrophoregrams with even denser and sharper bands than the 16-hr run.

Alkaline solutions of amino acids absorb carbon dioxide, giving carbamates, which may appear as additional spots or streaks after paper electrophoresis in alkaline electrolytes. Interference from carbamates is prevented by carrying out electrophoresis at 50°. Mixtures of arginine, histidine, citrulline, ornithine, and lysine are resolved by paper electrophoresis at 50° and pH 9.2 in an electrolyte containing sodium carbonate and sodium hydrogen carbonate. Addition of 8-hydroxyquinoline prevents interference from impurities in the paper.

Employing the technique of chromatography on paper prewetted with a buffer solution (Veronal-oxalate-citrate or phosphate), two distinct lipoprotein fractions were resolved from human serum. The developing solution consisted of a mixture of the buffer solution with isopropyl alcohol or ethanol. Since the necessity of prewetting the paper negates the possibility of reporting the chromatographic properties in terms of Rf values, Rb values were introduced. They represent the ratios of the distances moved by a given substance under study divided by the distance moved by a reference substance, bromophenol blue. The correlation between the lipoprotein fractions obtained by the paper chromatographic technique and those obtained from ultracentrifugal methods was established. On the chromatograms of the lipoproteins, the zone which moved farthest from the origin corresponded to the high-density lipoproteins, the α-lipoproteins. The α-lipoproteins as prepared by ultracentrifugal procedures separated into at least two well-defined spots. Resolution of these two α-lipoproteins by the paper chromatography of serum was not found. The spot closest to the origin corresponded to the low-density lipoproteins. The limited studies on the effect of alterations in the composition of the developing solvent indicated that resolution of the lipoproteins is probably due in a large part to their lipid composition. Prestaining of the serum gave essentially the same type of chromatographic results as those obtained by the poststaining technique. The paper chromatographic separation of serum lipoproteins was compared with the ionographic method. The ratio of β-lipoproteins to α-lipoproteins as determined by paper chromatography agreed well with the results obtained by ionography. Centrifugally accelerated paper chromatography was adapted to the separation of serum lipoproteins. The paper was prewetted with Veronal, Veronal-oxalate-citrate, or phosphate buffer solution and developed with mixtures of these buffers with isopropyl or ethyl alcohol. The radial type of development on the whole circular sheet caused the migrant to “fan out”, yielding developed spots of an arc-type shape, which made ordinary densitometric scanning difficult. By cutting “spokes” into the paper sheet, the fanning out of the migrant zones as they moved radially outward from the point of origin was circumvented. The technique was found to be rapid and practical, and yielded chromatograms, especially of serum prestained for lipoproteins, of high quality, which were readily scannable.

A method is described which permits the alkaline hydrolysis of oligoribonucleotides while on chromatography paper. In one example of this technique, the dinucleotides from a pancreatic RNase digest are characterized. In the second example, some new information about the diversity of sequences found at the 5′-triphosphate termini of bacterial RNA is obtained. There are at least three different pancreatic RNase fragments beginning with adenosine, two with guanosine, and at least three RNase T1 fragments beginning with adenosine.

Thiamine and its mono- and pyrophosphate esters (0–1 μg) were separated by paper chromatography, converted to their respective thiochromes, and measured fluorometrically. Excellent recoveries of added thiamine and thiamine phosphate from protein-containing mixtures were achieved by HCl extraction.

Two-dimensional chromatography on commercially available silica gel-loaded paper for the microanalysis of polar lipids from various tissues is described. All common phospholipids and their lyso derivatives can be reproducibly separated. As many as 22 lipid components were separated on a single chromatogram. Improved methods for staining lipids and for determining phosphorus in the chromatographic spots are reported.

Good resolution of aliphatic polyamines and other compounds related to the arginine pathway was obtained on paper strips impregnated with strong cation-exchange resin after chromatography with high molarity acidic buffers or hydrochloric acid. Quantitative estimation was achieved after eluting the spots with strong acid and dinitrophenylation. Recovery of acid-soluble pools from bacterial cells was monitored with radioactive markers which also served for spot location by scanning or autoradiography. These procedures share some of the advantages of paper and column chromatography.

1. (1) With the methods described, the oxidation of reduced glutathione GSH is prevented by addition of N -ethylmaleimide prior to precipitation of proteins. The oxidized glutathione (GSSG) is measured either enzymically with glutathione oxidoreductase or by paper chromatography. 2. (2) The true oxidized glutathione content of normal human red blood cells is 45 ± 10 μmoles/liter cells.

SUMMARY mitochondria were incubated in air in the presence and absence Methods are described for the resolution of coenzyme Q of substrates, the ultraviolet absorption spectra of the cyclo- homologues and other lipides by means of reversed phase paper hexane extracts of such particles were shown to change in a chromatography on silicone-impregnat,ed filter paper. manner which denoted a substrate-dependent reduction of coenzyme Q (9). Such extracts have now been chromatographed Acknowledgments-The authors are indebted to Dr. David on paper, and the results have been consistent with the previous E. Green for his encouragement in the course of these studies. interpretation of the spectra of these extracts. The data given Oscar Mayer and Company kindly supplied the large quantity in Fig. 2 indicate that the extract of particles incubated in the of tissue used in the investigation. REFERENCES

Methods for the separation of amino acids from tissue extracts by two-dimensional paper chromatography or by paper electrophoresis, have become well established in many laboratories. A number of one- and two-dimensional scanning devices have been developed for purposes of determining quantitatively the C14 radioactivity in amino acid spots on paper [1–4]. However the counting efficiency of these devices is low even when so-called 4π geometry is employed and both sides of the paper are counted simultaneously.

A filter paper assay was developed for measuring the activity of transamidating enzymes by the incorporation of radioactive amine substrates into proteins. The method is analogous to those generally employed for monitoring the enzymic coupling of isotopic groups to proteins, such as in the ATP-dependent phosphorylation of casein or histone. Use of minimal quantities of reagents, as well as simplicity of handling a large number of test samples, are distinct advantages of this technique. The guinea pig liver transglutaminase-catalyzed incorporation of C14-histamine and C14-putrescine into β-lactoglobulin and casein were studied. The apparent Michaelis kinetics of the reactions permitted evaluation of Km,app's for both isotopic substrates and competitive inhibition constants for nonradioactive amines were also obtained. In contrast to transglutaminase, the fibrinoligase—generated in human plasma from Factor XIII by the addition of thrombin—could not utilize β-lactoglobulin to an appreciable extent. Hence experiments had to be restricted to casein as acceptor protein. Apparent Michaelis constants for the isotopic substrates and inhibition constants for nonradioactive amines could be readily measured also in the complex plasma system. Finally, details of a rapid filter paper assay are described for measuring Factor XIII levels in human plasma by measuring the incorporation of C14-putrescine, at close to saturating concentrations, into casein.

A rapid chromatographic procedure was developed for the separation of sphingomyelin, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and free fatty acids on glass paper coated with sodium silicate. In addition, phosphatidyl choline and sphingomyelin were determined quantitatively by densitometry of the charred chromatogram, which was obtained by spraying the developed chromatogram with sulfuric acid and heating in an oven. The separation of phosphatides on sodium silicate-treated glass paper is more rapid than on silicic acid-impregnated paper, and the former is simpler to prepare. Preliminary application of this quantitative technique to human serum indicates that it may have a wide adaptability for the determination of phospholipids in natural products.

A highly specific method for qualitative and quantitative analysis of reducing carbohydrates has been described. The method involves the measurement of the amount of tritium found in [ 3 H]sodium borohydridereduced sugars following their paper chromatographic separation on ion-exchange papers. These procedures may be used to quantitate all of those components in 5 μl of a mixture which are present in amounts ranging from 0.1 to 10,000 nmoles. The quantitation gives approximately 10% accuracy at the lower end of the sensitivity range.

Pyrimidine and purine bases analysis by time of flight mass spectrometry and paper chromatography

Curves relating the R f of purines and pyrimidines on paper to the water content of acetonitrile-water mobile phases are used to predict the relative polarity of the solutes and the relative effect of functional groups in establishing these relative polarities. The water content of the mobile phase necessary to cause the solute to migrate to a given R f value is a measure of the polarity of the solute. Oxygen and amino groups enhance the polarity of purine, while methyl groups strongly decrease the polarity. At pH 10, oxygen is 2.5 times as effective as the amino group in establishing differences in polarity among the solutes.

A simple and specific chromatographic procedure for the quantitative determination of serum vitamin E is presented. Separation of vitamin E is accomplished by glass-fiber paper chromatography. Subsequently, vitamin E levels are estimated by a colorimetric procedure. The method was applied in a series of clinical experiments to illustrate the usefulness of this technique.

Two different two-dimensional decending paper chromatographic systems are presented which permit the simultaneous separation of the following: alanine, phenylalanine, tyrosine, glutamic, glucose, fructose, pyruvic, lactic, malic, fumaric, succinic, α-ketoglutaric, 3-phosphoglyceric, hexose phosphate, and phosphoenolpyruvic. Fructose diphosphate and 2-phosphoglyceric migrate together but can be well separated from the others. Carbon-14 labeled standards were used and localization accomplished by radioautography. The chromatography described can be applied usefully to deproteinated subcellular or tissue extracts from experiments in which isotopically labeled precursors were metabolized by the pathways of glycolysis, respiration, gluconeogenesis, or the biosynthesis and degradation of some amino acids. This is so chiefly because the discrete isolations obtained with permit the accurate determination of specific activities.

A previously described technique employing electrochromatography to separate amino acids rapidly has been modified. The use of DEAE ion-exchange paper to replace Whatman 3MM chromatography paper in the system permitted rapid separation of 20 amino acids instead of only 5 by the original method. The procedure has been applied for semiquantitative chromatography of amino acids from urine, plasma, and tissue extracts. After alkaline elution the individual amino acids can be quantitated spectrophotometrically.

The electrophoretic behaviour of oligodeoxynucleotides on ion-exchange papers was studied systematically with particular attention to applications in sequence analysis. Complex mixtures of larger oligonucleotides were fractionated by electrophoresis on cellulose acetate followed by t.l.c. on polyethyleneimine-cellulose. The behaviour of the oligonucleotides on polyethyleneimine-cellulose and on the two-dimensional system can provide a useful guide to their sequence. Detailed results from some of these experiments have been deposited as Supplementary Publication SUP 50031 (13 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

The instability of high energy bonds results in a gradual decomposition of nucleoside polyphosphates, even if they are stored dry at low temperatures. Therefore, they always contain detectable amounts of their less phosphorylated forms. On the other side, transphosphorylation reactions: 2 XDP → XTP + XMP and 2 XTP → XTetraP + XDP may take place in some specific conditions (1,2). Some preparations, if stored at wrong conditions, may be almost completely decomposed and may become the source of wrong and contradictory experimental results. Therefore, a simple and easy quantitative method should be available for control of purity of various nucleosidephosphate preparations stored in hundreds of laboratories, prior to their use.

A radiochemical method for the direct measurement of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) activity was described earlier (1,2). It avoided the difficulties associated with assay systems based on the coenzyme nature of thiamine pyrophosphate in TPP-dependent 1 enzyme reactions using apopyruvate decarboxylase (3) (2-oxoacid carboxylase, EC 4.1.1.1) or apotransketolase (4) (sedoheptulose-7-phosphate: d -glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1). Since the chromatographic isolation of TPP is time-consuming, a procedure for the rapid determination of thiamine pyrophosphokinase activity was desirable. The simplified method described here takes advantage of the anionic character of TPP. The assay is carried out with [ 14 C]thiamine as substrate. After incubation with the enzyme in the presence of Mg 2+ -ATP, the reaction mixture is applied to a DEAE-cellulose paper disc. The disc is extensively washed with sodium acetate resulting in the quantitative elution of [ 14 C]thiamine and partial retention of [ 14 C]TPP. This is quantitatively measured using the liquid scintillation counting technique. A similar procedure has been described for the determination of glycerol kinase (ATP: glycerol phosphotransferase, EC 2.7.1.30) and hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1) activities (5).

A new method especially adapted for the characterization of nucleoside diphosphate sugars in mixtures containing nucleoside mono-, di-, and tri-phosphates is described. It is based on ion-exchange chromatography using papers impregnated with polyethyleneimine (PEI). Nucleoside diphosphate sugars are separated from related compounds in 40 to 50 min. Good separations of the four common ribo- and deoxyribo-nucleoside monophosphate are achieved in about 4 hr. Thus homologous compounds are readily separated by this procedure which differ in the purine or pyrimidine component or which differ in the number of moles of phosphate present in the molecule.

A RADIOIMMUNOASSAY FOR HUMAN PITUITARY LUTEINISING HORMONE USING PAPER CHROMATO-ELECTROPHORESIS

Several electrophoretic and chromatographic systems are described for the separation of di- and tripeptides with N-terminal methionine. The most effective methods of separation are electrophoresis in barbital-triethylamine-acetic acid, pH 8.0 and chromatography in n -butanol-acetic acid-water, 18:2:5. Increased resolution may be obtained by using two or more systems in combination. These methods were intended to separate and identify the oligopeptides formed during the initiation of eukaryotic protein synthesis, but may also be applied to the separation of small peptides generally.

Two-dimensional paper chromatography was used to partition the acid-soluble phosphates in the erythrocytes of normal newborn infants, infants with erythroblastosis fetalis and adults. The inorganic phosphate concentration was higher in the fresh red cells of normal and erythroblastotic infants and rose more during incubation for four hours than in the cells of adults. The amount of erythrocyte adenosine-5’-triphosphate in the adults exceeded the quantities found in the cells of the two groups of babies. The 2,3-diphosphoglycerate level was greater in the erythrocytes of adults and infants with erythroblastosis than it was in the normal infants and incubation for four hours resulted in a sharper decrease of this compound in cells of the normal and erythroblastotic infants than in the adults.

A new paper electrophoretic method for the separation of bile acids into five groups, (1) unconjugated, (2) glycine conjugates and (3) taurine conjugates, and (4) and (5) the respective monosulfates, is described. Rapid and accurate qualitative and quantitative estimations of each group are obtained by densitometry after internal standardization and phosphomolybdate color development. The technique can be done in the routine clinical laboratory and is useful for the detection of diseases affecting the enterohepatic circulation of bile acids.

A method to exclude mites and other contaminants from test tube cultures is described. It consists in covering the mouths of the tubes with cigarette paper which is applied by pressing the flamed r...

In an attempt to elucidate the nature of ruthenium red reactions of substances in histochemical specimens, the effects of added electrolytes upon the reactions of model polyanions on filter paper and in tissue have been studied according to the Scott-Dorling type of critical electrolyte concentration (CEC) principle. The results obtained in the present experiments indicate that any of the three anionic groups, sulfates, phosphates and carboxyls can be responsible for the ruthenium red reactions of polyanions and that the dye binds to polyanions largely forming a salt type of linkage. However, ruthenium red is not a dye of choice for use in stainings based upon the CEC principle, inasmuch as certain moieties of the dye cations appear to interact with cationic electrolytes.

A sensitive, accurate, and precise method for the analysis of the separate sulfur-containing amino acids is described. Other amino acids do not interfere with the reaction, but certain solvents are unsuitable because of reagent bleaching. The method has been applied to the determination of the specific activity of amino acids separated by paper chromatography.

SUMMARY Chromatographic procedures for the detection and evaluation of oxygenated fatty acids are described. Emphasis has been placed on epoxy acids, but these methods promise to be of great value in studies of all classes of oxygenated acids. Paper chromatography of fatty acids and their esters has been developed for the examination of mixtures containing oxygenated derivatives. The method of adsorption chromatography on thin layers of silicic acid has been shown to be a powerful tool in studies of epoxy acids and hydroxy acids. Gas-liquid chromatography (GLC) of epoxy esters has been studied using both polar and nonpolar columns and has great utility in the detection and analysis of these compounds in mixtures. These methods have been applied to the examination of the epoxy components of six seed oils. Thin-layer and GLC proved particularly useful in this study and together demonstrated the presence of at least three distinct epoxy acids in each of these oils. Some conclusions as to the probable structures of these epoxy components are presented on the basis of their chromatographic characteristics in relation to known substances.