Isolation and analysis of a non-protein low molecular weight thiol-mercurial adduct from human prostate lymph node cells (LNCaP)

Abstract

Thiol compounds present in human malignant prostate cells (LNCaP) were investigated after reaction with a mercurial blocking reagent. After extracting the cellular glutathione and some other low molecular weight (LMW) thiols using trichloroacetic acid the resulting the protein precipitate was extracted with buffered 8 M urea containing 2-chloromercuri-4-nitrophenol in an equimolar amount to that of the thiol present. After removing the insoluble chromatin fraction the urea soluble labeled adducts formed were chromatographed on G15 Sephadex. Three yellow coloured (A410 nm) fractions were obtained; first, the excluded protein fraction containing 16.0 ± 4.1% of the applied label followed by an intermediate fraction containing 5.9 ± 1.2%. Finally a LMW fraction emerged which contained 77.2 ± 3.7% of the total label applied and this was further analyzed by column chromatography, first on an anion exchange column and then on a PhenylSepharose 6 column to give what appeared to be a single component. LC–MS analysis of this component gave a pattern of mercuri-clusters, formed on MS ionization showing possible parent ions at 704 or 588 m/z, the former indicating that a thiol fragment of molecular weight approximately 467 could be present. No fragments with a single sulfur adduct (a 369 m/z fragment) were observed The adduct was analyzed for cysteine and other amino acids, nucleic acid bases, ribose and deoxyribose sugars, selenium and phosphorus; all were negative leading to the conclusion that a new class of unknown LMW thiol is present concealed in the protein matrices of these cells.

Further Analytical Studies on a Mercuri Thiol Adduct Isolated from a Human Prostate Cancer Cell Line (LNCaP)

“…How-ever, it has been found to be present in high quantities in the cells studied. LNCaP cells contain 31.8 ± 2.2 femtomoles of Conthiol per cell constituting 56.5% of the total thiol present as opposed to their glutathione content of only 8.3 ± 0.7 femtomoles per cell (14.7%) [5].…”

“…Tests for ribose/pentoses using classic analytical techniques, for ribose using Bial's Orcinol method and for deoxyribose (2'-deoxypentose sugars) using the Dische Diphenylamine reagent, were negative [5].…”

“…All reagents and chemicals were of high analytical grade. Ellman reagent,(5, Full cysteine and other amino acid assays were carried on an ion exchange auto analyser by the Protein & Nucleic Acid chemistry facility of the Department of Biochemistry at the University of Cambridge.…”

“…In later work on the LNCaP cell line a thiol adduct of an unknown thiol, named a "Conthiol", was successfully prepared and isolated [5]. In this study 2-chloromercuri-4-nitrophenol (ClMNP-see supplementary information for details of preparation) was used as a thiol labelling agent to produce an adduct (RSMNP) as shown in Figure 1 below.…”