Determination of Covalently Bound Hypusine and Deoxyhypusine to Protein Using Submilligram of Protein Samples by HPLC

Abstract

A sensitive and reliable method for the determination of hypusine and deoxyhypusine in eIF-5A protein, an initiation factor of protein synthesis, was developed. An advantage of this method is the use of N^ε-(5-aminopentyl) lysine, an analogue of deoxyhypusine, as an internal standard. The application made it possible to determine hypusine in less than a mg of protein samples from cultured HTC cells and rat organs. After acid hydrolysis of protein samples to which had been added the internal standard, the hydrolysates were fractionated by carboxymethyl cellulose column chromatography. Also, diamine fractions containing a few pmol of hypusine and deoxyhypusine were successfully analyzed by a reversed phase HPLC with a fluorescence detection of o-phthalaldehyde. The method was applied for the determination of hypusine and deoxyhypusine in drug-treated HTC cells and normal rat organs. The results from HTC cells were discussed based on the known effects of each drug on hypusine biosynthesis.