A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. I. Stability of the mercury-sulfhydryl bond and influence of the ligand structure on immunochemical detection of the hapten.
The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic acid probes and their binding to sulfhydryl-hapten ligands, have been studied. Furthermore we developed a simple procedure for the preparation of mercurated probes at a microgram scale. Nucleic acids immobilized on Sephadex beads have been immunochemically detected after hybridization with mercurated probes and binding of the sulfhydryl-hapten ligand trinitrophenyl-glutathione. In this system, the method proved to be specific and sensitive. However, the same procedure, when applied in situ, failed to give a positive result. ELISA experiments showed that these results cannot be attributed to a suboptimal immunochemical detection of the ligand. Chromatographic analysis of mercurated polynucleotide-ligand complexes revealed, however, an unexpected lability of the mercury-sulfhydryl bond. Under non-equilibrium conditions, as present during a cytochemical washing procedure, the mercury-sulfhydryl b ond was found to dissociate rapidly. On basis of these results the hypothesis was forwarded that the bond between mercurated nucleic acids immobilized on Sephadex and the ligand was stabilized by the positive charge of the Sephadex matrix. This charge was introduced during the cyanogen bromide activation and inactivation necessary for the covalent coupling of nucleic acids to Sephadex. In situ, however, no such positive charges are present. By reversing the charge of the ligand we expected to stabilize the mercury-sulfhydryl bond. In a subsequent paper data are presented that confirm this hypothesis.
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Hopman
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Wiegant
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van Duijn
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003421:Crithidia / Q000235:genetics; D003911:Dextrans; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D006241:Haptens / Q000032:analysis; D006651:Histocytochemistry; D008024:Ligands; D008628:Mercury; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000032:analysis*; D013438:Sulfhydryl Compounds
D003911:Dextrans; D005456:Fluorescent Dyes; D006241:Haptens; D008024:Ligands; D009696:Nucleic Acids; D013438:Sulfhydryl Compounds; C025614:sephadex; D008628:Mercury