A three-step procedure for the isolation of peptides derived from adenine nucleotide binding sites of enzymes labeled with p-fluorosulfonyl [14C]benzoyl-5′-adenosine
✍ Scribed by Fred S. Esch; William S. Allison
- Publisher
- Elsevier Science
- Year
- 1979
- Tongue
- English
- Weight
- 334 KB
- Volume
- 95
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A simple three-step procedure is described for the purification of a labeled peptide from a tryptic digest of the P-subunit of the F,-ATPase after the enzyme had been inactivated with p-fluorosulfonyl-[14C]benzoyl-5'-adenosine.
The procedure involves: (1) anion-exchange chromatography of a tryptic digest of the labeled p-subunit on diethylaminoethyl-Sephadex; (2) treatment of the peptides in the radioactive peak from the first step with 0.1 M NaOH under conditions in which the ester bond in the label is hydrolyzed; and
(3) anion-exchange chromatography of the treated peptides under conditions identical to those of the first step after removal of the NaOH by gel filtration. Cleavage of the ester bond in the second step releases adenosine and specifically introduces an additional negative charge onto the labeled peptide. Thus, it is resolved from the peptides that contaminate it in the third step.