Alkaline phosphatase electrophoresis paper

W12. Wolfson, W. Q., Location of alpha-2 globulin by demonstration of alkaline phosphatase during paper electrophoresis. Nature 180, 550-551 (1957). 

In this paper we report the assaying of individual molecules of the enzyme alkaline phosphatase (EC 3.1.3.1) by capillary electrophoresis (CE) utilizing laser-... 

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. 

In Ghosh and Nath s experiments with paper electrophoresis (barbitone buffer, pH 8.6), rachitic serum showed an alkaline phosphatase mobility close to the mobility of jS-globulin (Nil). Sera from infective hepatitis and obstructive biliary cirrhosis, however, showed maximum alkaline phosphatase activity in the 2-globulin zone (G5). 

Electrophoresis on cellulose acetate paper has also been employed for the characterization of human alkaline phosphatase isoenzymes by Korner (K20, K21) and Posen et al. (P19). A modification of the Gomori technique (G11-G13) for the histochemical localization of alkaline phosphatase was made by Allen and Hyncik (AlO) to visualize the enzyme zones in the starch and agar gels. 

G7. Ghosh, N. K., and Nath, R. L., Paper electrophoresis of human serum alkaline phosphatase in hepatic disorders. Proc. Combined 61st and BSnd Indian Sci. Congr., CcdcnMa, Part III, Sect. XI (Physiology), pp. 595-596 (1964-1965). 

The inactivated butirosin A darkens at 217-221°. The empirical formula of butirosin A monophosphate monohydrate was determined by elementary analysis. It gave positive ninhydrin, Rydon-Smith, and Hanes reactions. On high-voltage, paper electrophoresis under 3.5 kV for 15 minutes with 1 3 36 (v/v) formic acid-acetic add-water, the inactivated butirosin A moved 14.6 cm towards the cathode, whereas butirosin A moved 19.0 cm. It showed only end absorption in its u.v. spectrum, and a band at 960 cm (phosphoric ester) in the ij. spectrum. Hydrolysis by alkaline phosphatase yielded butirosin A. 

General. Pyridine is distilled from p-toluenesulfonyl chloride and then from calcium hydride and stored over potassium hydroxide. N,N-Di-methylformamide is distilled from calcium hydride and stored over molecular sieves. Dioxane is distilled from phosphorus pentoxide. Tri-n-butylamine, triethylamine, diphenyl phosphorochloridate, and o- and p-fluorobenzoyl chlorides are distilled before use. Tri-n-butylammonium pyrophosphate is prepared at room temperature according to the method of Moffatt and Khorana and stored at 5°. Paper chromatography is carried out by the descending technique on Whatman No. 1 paper in (A) isobutyric acid-1 M NH4OH (60 40) and (B) 1-propanol-water (7 3). Electrophoresis is carried out on Whatman No. 1 paper at pH 3.5 in 0.035 M citric acid-0.0148 M sodium citrate (1 1). Evaporations are carried out under reduced pressure at bath temperatures below 30°. Phosphate analyses of nucleoside triphosphates are performed by the method of Lowry and Lopez after treatment of approximately 1 / mole of these compounds for 60 min at 22° in 1 ml of Tris chloride at pH 10.4 containing 0.02 mg of alkaline phosphatase of calf intestinal mucosa (type VII, Sigma Chemical Co.). 

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