ISOLATION AND HPLC OF N-EPSILON-LITHOCHOLYL LYSINE AS ITS FLUORESCAMINE AND DIMETHYLAMINOAZOBENZENE ISOTHIOCYANATE DERIVATIVES

N-epsilon-lithocholyl lysine (NELL) is a component of tissue-bound lit hocholic acid (TBL). The isolation of NELL from native protein sources was simulated by hydrolysis of lithocholyl-bovine serum alburmin (BSA ) (synthesized by coupling lithocholyl-N-hydroxysuccinimide to fatty a cid-free BSA) by digestion with a mixture of 6N HCl-propionic acid at 70 C for 3 h under partial vacuum. NELL was isolated on a reversed pha se Sep-Pak C-18 column and converted to either a fluorophor with fluor escamine or to a chromophor with dimethylaminoazobenzene isothiocyanat e for subsequent HPLC using appropriate fluorescence or UV/visible abs orption detectors. The procedure described here is quantitative, highl y sensitive, and not dependent upon the use of Clostridial choranoylam ino acid hydrolase, the activity of which is sometimes blocked by ster ic hindrance on the substrate. Using this procedure we have demonstrat ed the presence of TBL in native histones.