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Isolation and HPLC of N-ε-lithocholyl lysine as its fluorescamine and dimethylaminoazobenzene isothiocyanate derivatives

Authors
Publisher
Elsevier Inc.
Publication Date
Volume
59
Issue
3
Identifiers
DOI: 10.1016/0039-128x(94)90030-2
Keywords
  • Lithocholic Acid
  • Tissue-Bound Lithocholic Acid
  • Bile Acid Conjgate
  • Bile Acid
Disciplines
  • Biology

Abstract

Abstract N-ε-lithocholyl lysine (NELL) is a component of tissue-bound lithocholic acid (TBL). The isolation of NELL from native protein sources was simulated by hydrolysis of lithocholyl-bovine serum albumin (BSA) (synthesized by coupling lithocholyl-N-hydroxysuccinimide to fatty acid-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 phase Sep-Pak C 18 column and converted to either a fluorophor with fluorescamine or to a chromophor with dimethylaminoazobenzene isothiocyanate for subsequent HPLC using appropriate fluorescence or UV/visible absorption detectors. The procedure described here is quantitative, highly sensitive, and not dependent upon the use of Clostridial cholanoylamino acid hydrolase, the activity of which is sometimes blocked by steric hindrance on the substrate. Using this procedure we have demonstrated the presence of TBL in native histomes.

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