Electrophoresis, on paper

Jin and Zhou have determined 4-aminobenzoic acid in procaine injection solutions by electrophoresis on paper [147]. A 5 pL portion of the injection solution was diluted to 2 mg/mL in procaine hydrochloride, and applied to No. 1 paper (25 cm x 24 cm) for electrophoresis in a JMDY-WI apparatus. The system used a pH 3 buffer solution (9.76 g of citric acid and 1.03 g of sodium citrate in 100 mL), and a potential gradient of 20 V/cm applied for 20 minutes. The paper was then dried and sprayed with a solution containing 100 mL of ethanolic 2% / -dimethylaminobenz-aldehyde and 5 mL of anhydrous acetic acid. A standard solution containing 0.03 pg/pL of 4-aminobenzoic acid was used for comparison. 

Fig. 1. Effect of pH on the hydrolysis of mixed-base 3 -phosphorylated ribodinucleo-tides (O) and deoxyribodinucleotides (A). For ribonucleotides reaction mixtures (total volume 0.063 ml) were incubated for 1 hr at 37° in sealed capillary tubes. Substrate Am = 65, [enzyme] = 4.2 units/ml, [buffer] =0.1 M (tris succinate), [MgCUl = 1.0 mM. After electrophoresis on paper for 0.5 hr, 23 V/cm, pH 7.0, amounts of N were determined as percent of total materia) (A271) recovered from the paper. With deoxyribodinucleotides [substrate] = 83 Am, [enzyme] = 5.1 units/ml [buffer] and [MgCl2] as above. Reprinted from Richards and Laskowski (SO). Copyright (1969) by the American Chemical Society. Reprinted by permission of the copyright owner.

Owen, J. A., Determination of serum-protein fractions by zone electrophoresis on paper and direct reflection photometry. Analyst 81, 26 (1956). 

R5. Rosenberg, I. N., Serum lipids studied by electrophoresis on paper. Proc. Soc. Exptl. Biol. Med. 80, 751 (1952). 

Using the technique of electrophoresis on paper, Forfar et al. (F7) found that cholesterol was abnormally concentrated in the (3-lipoprotein fraction. Using reverse phase chromatography, an attempt was made to demonstrate sterols other than cholesterol, but this was unsuccessful. If they are actually present, such substances must have an Rf similar to that of cholesterol or else be present in quantities too small to be demonstrated by the technique employed. 

Most important for successful assays is the method of separating bound from free ligand. In the original study of Yalow and Berson using human anti-insulin, this was accomplished by electrophoresis on paper. 

This material, described by Nakahara and Fukuoka (Nl), is present only in cancer gastric juice. Upon intraperitoneal injection into mice, it depresses the catalase titer of the liver, kidney, and blood. The relationship of this material to mucopolysaccharides and mucoproteins of gastric juice is not well known. Toxohormone— like material obtained from normal stomach which is biologically inactive, however—was found to have lower polarographic activity than that from stomachs with gastric cancer (M18). Separation of materials with toxohormone activity by continuous electrophoresis on paper curtain and column chromatography is discussed in the following review in this volume. 

Takamura et al. and Wada et gastric juice of normal individuals and patients with histamine-fast anacidity and gastric carcinoma by continuous electrophoresis on paper curtain, and assayed the chemical composition and biological activities of the fractions obtained. Electrophoresis was performed in veronal buffer of pH 8.6 and ionic strength of 0.03, at 225 volts and 7.5-8.25 mA for 24 hours. As in paper strip electrophoresis, 4 major components were obtained, named by the authors (from anode to cathode) Bl, B2, B3, and B4 B3 contained three minor components A, B, and C. In addition, acidic gastric... 

In studies performed in association with J. A. Buckwalter s group from Iowa City, we studied the distribution of ABO(H) blood group substances in the electrophoretic partition of gastric juice (G3a, G6). Several acid and anacid pools of gastric juice were subjected to continuous electrophoresis on paper curtain in borate buffer of pH 9.0, 0.06. The... 

Continuous electrophoresis on paper curtain was applied in our laboratory in fractionating intrinsic factor activity of human gastric juice (G20). In each run, 30-100 mg of dialyzed and lyophilized gastric juice dissolved in borate buffer of pH 9.2 or acetate buffer of pH 4.0 (0.15 and 0.1 ionic... 

G20. Glass, G. B. J., Stephanson, L., Rich, M., and Laughton, R. W., Intrinsic-factor activity of human gastric juice after fractionation by continuous electrophoresis on paper curtain. Brit. J. Haematol. 3, 410-411 (1957). 

K8. Kubo, K., Castro-Curel, Z., Ibanez, N., Glass, G. B. J., and Gode, G. F., Fractionation of gastrone, inhibitory material to gastric secretion by continuous electrophoresis on paper curtain, column chromatography and gel filtration. Gastroenterology 46, 748 (1964). Abstr. 

Preece and Hobkirk have attempted the fractionation of water-soluble, cereal polysaccharides (obtained from rye and oats), using zone electrophoresis on paper in a borate buffer at pH 11 and an acetate buffer at pH 4. The fractions were located on the pherogram by segmenting it, eluting... 

Smith (S37) has stated that electrophoresis is a suitable procedure (for amino acid separations) as it is quick, it avoids the need to desalt and is also applicable to whole blood and urine. High voltage electrophoresis on paper has been extensively used for the separation of amino acids and peptides in biological fluids (A9, ElO, F7, H12, Jll, K2, Nl, P18). Review articles on high voltage electrophoresis have been published by Griddle et al. (C15), Blackburn (B16), Efron (ElO), Grassini (G23), and Ritschard (R3). 

In this laboratory, attempts (G6, G8) have been made to purify and crystallize human placental alkaline phosphatase enzyme by a number of procedures involving homogenization with 0.05 M Tris buffer (pH 8.6), extraction with butanol, ammonium sulfate precipitation, exposure to heat, ammonium sulfate fractionation, dialysis, repeated ethanol fractionation, gel filtration with Sephadex G-200 (Fig. 18), continuous curtain electrophoresis on paper (Beckman Model CP), multiple TEAE-cellulose anion exchange chromatography, and equilibrium dialysis. Variant A (electrophoretically fast-moving) of human placental alkaline... 

We will now deal separately with the electrophoresis on paper, starch gel, agar gel, and Sephadex gel of alkaline phosphatase. 

The world of electromigration separations is sharply divided into two areas. Zone electrophoresis on paper and related procedures have (in spite of their wide applicability to diverse organic compounds) already passed their period of favour. The other branch is represented by the more recent techniques some of which have already became widely accepted (such as isoelectric focusing or separations in polyacrylamide gel) and the others that are at the moment in the centre of a rapid development like displacement electrophoresis (isotachophoresis). This chapter is devoted mainly to analytical procedures such as these which are governing the area of electromigration separations at the moment with a single exception flow deviation (curtain) electrophoresis which will be discussed in more detail because it offers several new dimensions in the separation field. The other preparative procedures are summarized only briefly. 

Wieland Theodor bora in 1913 in Munich, he studied chemistry in Freiburg Brsg. and Munich where he received his Dr. phil. in 1937. From 1937 until 1946 he was assistant, (Lecturer 1941), with Richard Kuhn at the Kaiser-Wilhelm/Max-Planck-Institute in Heidelberg. In 1946 he became Prof, at the Univ. of Mainz. Between 1951 and 1968 he was Prof, and Director of the Institute of Organic Chemistry at the Univ. of Frankfurt. After R. Kuhn s death in 1967, he was appointed Director of the Department of Chemistry, later Natural Products, of the Heidelberg Institute until 1981. He is hon. prof, of the Universities of Frankfurt and Heidelberg. His contributions include electrophoresis on paper of amino acids, peptides and proteins, isoenzymes of lactate dehydrogenase (with G. Pfleiderer), mixed anhydride method of peptide synthesis (p. 79) peptides of Amanita mushroonis (p. 211). 

Electrophoresis on paper using a single buffer was applied to bile acid separation by Biserte et al. (41). The buffer system was made up of 30 ml of pyridine and 100 ml of glacial acetic acid in 5 liters of water. The pW was 3.9. In this system, cholic, glycocholic, taurocholic, and taurochenodeoxycholic acids were attracted toward the anode at rates increasing in the order named. Curiously, free deoxycholic acid remained at the origin. The locations of the bile acids on the paper were found by spraying with phosphomolybdic acid. Results with various animal biles were reported. 

Both standard acids and conjugates from natural sources have been resolved by Briggs et al. (40) and Biserte et al. (41) using electrophoresis on paper, and the latter authors have reported applications to the analysis of the acids in animal biles. Norman (42) used paper electrophoresis to study the bile acids in the gallbladder bile and hepatic bile of man. 

Schilling and Deiss (1953) added radioactive Bu to gastric juice prior to electrophoresis on paper. The radioactive B12 was bound to a component of gastric juice which was not one of the major protein peaks and which moved towards the anode. 

During electrophoresis on paper B12 may remain attached (bound) to a protein and move with it, e.g., towards the anode, or otherwise in a manner different from unbound B12 (Latner et al., 1952, 1953 Schilling and Deiss, 1953 Pitney et al., 1954, 1955). 

After the first fractionation with ammonium sulfate, the solution appeared to be homogenous in electrophoresis on paper. However, moving boundary electrophoresis and ultracentrifugation showed that there were in reality four major components present. After a second fractionation with ammonium sulfate, a single peak was obtained, but it was strongly asymmetric. After a rapid fractionation with acetone in the presence of calcium acetate, the dissymmetry of the peak disappeared completely and the protein appeared to be homogenous by moving boundary electrophoresis also. 

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