The isolation of phosphoarginine

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Abstract
Tail muscle was dissected from live crayfish and dropped into liquid N2. The frozen muscle was extracted with 9% trichloracetic acid and filtered into 10 [image] NaOH. HC1 (5[image]) was added to pH 9.0 and barium acetate added until no further precipitation occurred and the pH readjusted. The soluble Ba salts in the filtrate were precipitated with 3 volume of 95% ethanol at pH 9, collected, washed and dried. The phosphoarginine (PA) was then pre cipitated as a Cu salt by dissolving 5 g in 150 ml water with 5 [image] HC1, adjusting the pH to 9.0, removing the barium phosphate, adding CuCl2 solution (1 g/ml) and adjusting the pH to 6.5. The Cu precipitates were suspended in 100 ml water, 12 ml of 5 [image] HC1 and 0.5 ml of CuCl2 added, shaken, neutralized with 5 [image] NaOH, pH adjusted to 6 5,centrifuged and the washing repeated until free of Ba. The Cu was then removed with H2S, the solution decolorized with charcoal, and 9 volumes of ethanol added to the solution at pH 3.5. The pH was then adjusted to S.O to give the hydrochloride. This was precipitated twice, as above but with 1.5 volumes of ethanol. The PA was then precipitated as the Ba salt by adding barium acetate at pH 9.0, adjusting to pH 8.2 and adding 2 volumes of alcohol. The yield of pure Ba salt was 10 gAg (49%). The losses (46%) in preparing the Cu salt and hydrochloride were removed by precipitating as the Ba salt. All steps were conducted in the cold.