Using periodate-oxidized nucleotide as affinity label for the nucleotide site of proteins

Abstract

The active site of pigeon liver malic enzyme was labeled with a fluorescent affinity label, the periodate-oxidized aminopyridine adenine dinucleotide phosphate. The modified enzyme was subjected to proteolytic digestion with trypsin. The resulted peptides were then separated with reversed-phase high-performance liquid chromatography on Waters muBondapak C18 column. Two pure fluorescent peptides were obtained after three runs of the chromatography. The peptides were then subjected to automatic Edman degradation on a Beckman peptide sequencer and subsequently separated and identified with phenylthiohydantoin C18 column. No sequence was obtained. The possible reasons for the failure in sequencing the periodate-oxidized nucleotides labeled active site peptide and some possible pitfalls in using these reagents were discussed.