Analysis of Low Molecular Weight Thiols Present in a Human Prostate Cancer Cell Line (LNCap) Using 4- Dimethylaminoazobenzene-4'-Maleimide (DABMA) as a Labeling Agent: Apparent Glutathione Content
International Journal of Sciences, Issue 11, 2012
17 Pages Posted: 7 Jul 2017
Date Written: November 12, 2012
Low molecular weight thiols play important roles in cell homeostasis, drug metabolism, cancer formation and progression. The major component in this fraction is commonly assumed to be the tripeptide glutathione but there have been reports of the presence of other unknown thiols in tumour cells. To investigate this further the composition of the non protein sulphydryl components of the acid soluble fraction (ASF) of LNCap cells (a lymph node human prostate cancer) have been investigated after labelling with 4- dimethylaminoazobenzene-4’-maleimide (DABMA) at pH 5.5.
A preliminary isolation of the adducts formed was achieved using reverse phase chromatography on C-18 silica gel (ODS-AQ) giving three crude fractions. The first fraction contained the glutathione adduct and this could be separated from the other thiol adducts by flash chromatography on silica gel followed by ion exchange chromatography on a cellulose anion exchanger.
The molar extinction coefficient of the glutathione derivative at the λ max of 455nm (pH5.5) was estimated by amino acid analysis to be 26,600 M-1 cm-1 (DABMA in methanol 32,000 M-1 cm-1 ). Using this value it was calculated that, in the total ASF, not more than 40% of the total measured thiol could be attributed to glutathione. Other thiol adducts were shown to be present in variable amounts; separation and analysis proved difficult but using HPLC and TLC techniques a number of unknown components were shown to be present. Amino acid analysis revealed that they did not contain cysteine or peptide material and UV/visible spectra ruled out the presence of nucleic acid derivatives. MS and NMR analyses indicated that unknown water soluble thiols are present which are probably lipid in nature. Further analysis of one isolated adduct indicated the presence of 5-mercapto-pentanol.
Keywords: Thiols, Acid Soluble Fraction, Glutathione, Prostate Cancer Cells, Analysis, Maleimide Adducts, Silica Gel Chromatography, HPLC, TLC
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