Abstract
A method for determination of diadenosine tetraphosphate, Ap4A, by high pressure liquid chromatography has been developed. The free nucleotides were extracted from biological material with 10% trichloroacetic acid, which subsequently was removed with n-trioctylamine dissolved in freon; the aquous phase containing the nucleotides was lyophilized. The lyophilized material was dissolved in 0.1m-NaHCO3, digested with alkaline phosphatase, injected into a strong anion exchange column without adjustment of pH and chromatographed isocratically with a KH2PO4−KCl buffer at pH 4.3. The enzymatic digestion of the cellular nucleoside mono-, di-, and triphosphates was followed and found to be completed in two hours, while Ap4A was observed not to be cleaved during this period. Amounts as small as 5–10 picomoles of Ap4A were determined quantitatively by the method described.
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