Determination of epsilon (gamma-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation.
A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the epsilon (gamma-glutamyl)lysine isopeptide bond, which is usually formed by protein-crosslinking transglutaminases between polypeptide chains. The procedure is based on the separation and quantitation of epsilon (gamma-glutamyl)lysine isodipeptide following exhaustive proteolytic digestion of the crosslinked peptide. It involves preliminary separation steps on a cation exchanger resin and a silica HPLC column, precolumn derivatization with phenylisothiocyanate, and reversed-phase high-pressure liquid chromatographic separation on a C18 column. The derivatized isodipeptide gave a linear concentration-response relationship, with a detection limit of 10 pmol/mg of protein. The combination of the preliminary separation steps and the sensitive detection system permits the determination of the epsilon (gamma-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.
Department of Biochemistry, University School of Medicine, Debrecen, Hungary.
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Tarcsa
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Fesus
D001792:Blood Platelets / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003432:Cross-Linking Reagents; D006801:Humans; D006868:Hydrolysis; D007474:Ion Exchange; D017879:Isothiocyanates; D009994:Osmolar Concentration; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013861:Thiocyanates
D003432:Cross-Linking Reagents; D017879:Isothiocyanates; D013861:Thiocyanates; C005441:phenylisothiocyanate