Determination of the unusual amino acid hypusine at the lower picomole level by derivatization with 4-dimethylaminoazobenzene-4'-sulphonyl 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 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 determine the correct positioning of the hypusine residue in the amino acid sequence, which was not possible by the amino acid sequencing procedure alone.
Bartig
D
D
Institut für Biochemie, Universität Kiel, Germany.
Klink
F
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D007202:Indicators and Reagents; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet; D016945:Sulfolobus acidocaldarius / Q000378:metabolism; D014357:Trypsin; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives* / Q000737:chemistry
D001426:Bacterial Proteins; D007202:Indicators and Reagents; C100028:hypusine; C010572:4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride; D004124:p-Dimethylaminoazobenzene; D014357:Trypsin; D008239:Lysine