DETERMINATION OF THE UNUSUAL AMINO-ACID HYPUSINE AT THE LOWER PICOMOLE LEVEL BY DERIVATIZATION WITH 4-DIMETHYLAMINOAZOBENZENE-4'-SULFONYL CHLORIDE AND REVERSED-PHASE HIGH-PERFORMANCE OR MEDIUM-PRESSURE LIQUID-CHROMATOGRAPHY

Hypusine, an unusual amino acid formed by post-translational modification of lysine, is normally determined by specific metabolic labelling followed by measurement of released radioactivity after protein hydrolysis. This paper describes a sensitive non-radioactive method for the determination of hypusine, involving complete protein hydrolysis and precolumn derivatization of the released amino acids with 4-dimethylaminoazobenzene-4'-sulphonyl chloride, followed by reversed-phase high-performance or medium-pressure liquid chromatography of the dabsylated derivatives. The detection limit of hypusine was about 500 fmol. Additionally, the hypusine-containing containing protein from the archaebacterium Sulfolobus acidocaldarius was purified. By applying the dabsylation method to the analysis of tryptic peptides derived from this protein, it was possible to deter-mine the correct positioning of the hypusine residue in the amino acid sequence, which was not possible by the amino acid sequencing procedure alone.